C-terminal GFP Research Articles

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

BackgroundExtracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications.MethodsIn this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors.ResultsFusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies.ConclusionsThis study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.

A nanobody-based strategy for rapid and scalable purification of human protein complexes.

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.

Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron.

Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.

Localization and characterization θ carbonic anhydrases in Thalassiosira pseudonana.

Carbonic anhydrase (CA) is a crucial component for the operation of CO2-concentrating mechanisms (CCMs) in the majority of aquatic photoautotrophs that maintain the global primary production. In the genome of the centric marine diatom, Thalassiosira pseudonana, there are four putative gene sequences that encode θ-type CA, which was a type of CA recently identified in marine diatoms and green algae. In the present study, specific subcellular locations of four θCAs, TpθCA1, TpθCA2, TpθCA3, and TpθCA4 were determined by expressing GFP-fused proteins of these TpθCAs in T. pseudonana. As a result, C-terminal GFP fusion proteins of TpθCA1, TpθCA2, and TpθCA3 were all localized in the chloroplast; TpθCA2 was at the central chloroplast area, and the other two TpθCAs were throughout the chloroplast. Immunogold-labeling transmission electron microscopy was further performed for the transformants expressing TpθCA1:GFP and TpθCA2:GFP with anti-GFP-monoclonal antibody. TpθCA1:GFP was localized in the free stroma area, including the peripheral pyrenoid area. TpθCA2:GFP was clearly located as a lined distribution at the central part of the pyrenoid structure, which was most likely the pyrenoid-penetrating thylakoid. Considering the presence of the sequence encoding the N-terminal thylakoid-targeting domain in the TpθCA2 gene, this localization was likely the lumen of the pyrenoid-penetrating thylakoid. On the other hand, TpθCA4:GFP was localized in the cytoplasm. Transcript analysis of these TpθCAs revealed that TpθCA2 and TpθCA3 were upregulated in atmospheric CO2 (0.04% CO2, LC) levels, while TpθCA1 and TpθCA4 were highly induced under 1% CO2 (HC) condition. The genome-editing knockout (KO) of TpθCA1, by CRISPR/Cas9 nickase, gave asilent phenotype in T. pseudonana under LC-HC conditions, which was in sharp agreement with the case of the previously reported TpθCA3 KO. In sharp contrast, TpθCA2 KO is so far unsuccessful, suggesting a housekeeping role of TpθCA2. The silent phenotype of KO strains of stromal CAs suggests that TpαCA1, TpθCA1, and TpθCA3 may have functional redundancy, but different transcript regulations in response to CO2 of these stromal CAs suggest in part their independent roles.

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting-heads motif and super-relaxed state of myosin.

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

SUMMARYSerine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Insights into the role of N‐linked glycosylation in directing plasma membrane localization of GPCRs expressed in Saccharomyces cerevisiae

Glycosylation is the biochemical process of the attachment of sugar moieties or carbohydrates to proteins during co‐translational or post‐translational modification (PTM). It is one of many types of protein modifications that naturally occurs during the process of protein expression in organisms like Saccharomyces cerevisiae (Baker’s yeast). For natively expressed proteins, glycosylation can be critical for proper protein processing and can also be important for the production of proteins in a stable and biologically active state. For proteins that are non‐natively, or heterologously, expressed in these organisms, glycosylation can lead to improper processing and ultimately produce proteins with little to no biological relevance. This proves to be an undesirable effect for the elucidation of protein structure. However, large quantities of protein are required for structural analysis, presenting a significant obstacle for structural biologists and those who use heterologous expression as a means to acquire large quantities of protein. This problem becomes significantly more challenging for membrane proteins.Membrane proteins are an elusive class of biomolecules that reside or closely associate with the lipid bilayers of cells and encompass transporters, receptors and channel pores, to name a few. They play an intrinsic role in maintaining proper cell function including signaling and selective uptake of extracellular ions and small molecules. G‐protein coupled receptors (GPCRs) constitute one of the largest groups of membrane proteins across the plant and animal kingdoms. Despite being of scientific and medical interest, GPCRs and many integral membrane proteins are notoriously difficult to isolate and work with because of their hydrophobicity, structural flexibility and instability in the absence of the cell membrane. Optimized experimental conditions often vary from protein to protein and necessitate a systematic approach to achieve. This typically requires labor and resource intensive efforts. The goal of this work is to uncover new insights into the role of post‐translational processing and protein localization in the cell. Using a combination of fluorescence microscopy and bioinformatics we formulated an approach to investigate the effects of N‐linked glycosylation on plasma membrane localization of GPCRs expressed in S. cerevisiae. We postulate a critical link between plasma membrane localization and expression of “ligand active” or structurally relevant GPCRs. Previous studies of the human adenosine A2A receptor (hA2AR) show it readily localizes to the plasma membrane during expression in yeast, while the human dopamine D2 receptor (hD2R) does not. A sequence comparison of the two reveals three putative N‐linked glycosylation sites at the N‐terminal extracellular region of hD2R which is lacking in hA2AR. Using a chimera of these two receptors prepared with a C‐terminal GFP tag, we show the chimera does not traffic to the plasma membrane similarly to full‐length hA2AR, suggesting N‐linked glycosylation strongly influences functional high‐level expression of GPCRs and possibly other membrane proteins in yeast.

Siamese Cat Tyrosinase has Enhanced Proteasome Degradation and Increased Cellular Aggregation

Aim: Siamese cats are a notable example of a temperature-sensitive partial albinism phenotype. The signature color-pointing pattern is the result of an amino acid substitution - G302R - in the cysteine-rich domain of feline tyrosinase. The precise mechanism for the loss of tyrosinase enzyme activity due to this mutation is unknown. Objective: We have used a cellular biology approach to unraveling the relationships between feline coloration, behavior and increased risk for feline cognitive dysfunction syndrome. GFP-fusion constructs of wild type domestic short hair tyrosinase and Siamese (G302R) tyrosinase were generated to study cellular trafficking, degradation and the propensity for cellular aggregation. Data Description: C-terminal GFP G302R expression has reduced Golgi localization, increased cytosolic fractions with reduced calnexin co-localization. N-terminal GFP constructs were retained in the ER, with little to no Golgi associated forms. C-terminal and N-terminal GFP G302R TYR is observed to have increased high molecular weight aggregation following proteasome inhibition.

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting heads motif and super-relaxed state of myosin

Formation of the auto-inhibited super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We found that a single point mutation, E525K, associated with dilated cardiomyopathy (DCM) dramatically enhances the stability of the SRX state. We expressed human cardiac myosin containing the subfragment 1 domain, 15 heptads of the subfragment 2 coiled-coil region and a leucine zipper that facilitates dimerization, as well as a C-terminal GFP tag (M2β 15HPZ.GFP).

Transcriptome analysis of Leucojum aestivum and identification of genes involved in norbelladine biosynthesis.

Transcriptome analysis of Leucojum aestivum led to the identification of 50 key genes associated with Amaryllidaceae alkaloid biosynthesis including norbelladine synthase which localized in the cytosol and catalyzed norbelladine formation. The Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites, which are known for their biological activities. Although the general chemical reactions in the AA biosynthetic pathway have been proposed, the genes and enzymes of the pathway remain largely unstudied. All AAs are synthesized from a common precursor, norbelladine, by the condensation of tyramine and 3,4-dihydroxybenzaldehyde. The enzyme norbelladine synthase (NBS) which catalyzes the condensation reaction has only been characterized at a molecular level from one species, and the subcellular localizations have not been explored. Hence, the intracellular compartments wherein the AAs are biosynthesized remain unknown. In this study, a first comprehensive transcriptomic analysis of summer snowflake (Leucojum aestivum) was done to identify key genes associated with AA biosynthesis. Fifty orthologous genes were identified and deposited into GenBank. In addition, we identified and further characterized NBS from the transcriptome of L. aestivum and previously reported Narcissus papyraceus. Phylogenetic analysis showed that LaNBS, NpNBS1 and NpNBS2 shared high amino acid identity. The heterologous expression of LaNBS produced a recombinant protein with NBS activity. Bioinformatic prediction and C-terminal GFP tagging in transiently transformed Nicotiana benthamiana showed that LaNBS, NpNBS1 and NpNBS2 were likely localized to the cytosol which suggests that the AA biosynthesis starts in the cytosol. This study provides an Amaryllidaceae transcriptome that will be very helpful to identify genes for characterization studies in AA metabolism in planta or using heterologous systems. In addition, our study will facilitate the bioengineering of AA biosynthetic pathway in plants or in microorganisms.

MtNPF6.5 mediates chloride uptake and nitrate preference in Medicago roots.

The preference for nitrate over chloride through regulation of transporters is a fundamental feature of plant ion homeostasis. We show that Medicago truncatula MtNPF6.5, an ortholog of Arabidopsis thaliana AtNPF6.3/NRT1.1, can mediate nitrate and chloride uptake in Xenopus oocytes but is chloride selective and that its close homologue, MtNPF6.7, can transport nitrate and chloride but is nitrate selective. The MtNPF6.5 mutant showed greatly reduced chloride content relative to wild type, and MtNPF6.5 expression was repressed by high chloride, indicating a primary role for MtNPF6.5 in root chloride uptake. MtNPF6.5 and MtNPF6.7 were repressed and induced by nitrate, respectively, and these responses required the transcription factor MtNLP1. Moreover, loss of MtNLP1 prevented the rapid switch from chloride to nitrate as the main anion in nitrate-starved plants after nitrate provision, providing insight into the underlying mechanism for nitrate preference. Sequence analysis revealed three sub-types of AtNPF6.3 orthologs based on their predicted substrate-binding residues: A (chloride selective), B (nitrate selective), and C (legume specific). The absence of B-type AtNPF6.3 homologues in early diverged plant lineages suggests that they evolved from a chloride-selective MtNPF6.5-like protein.

MtCOPT2 is a Cu+ transporter specifically expressed in Medicago truncatula mycorrhizal roots.

Arbuscular mycorrhizal fungi are critical participants in plant nutrition in natural ecosystems and in sustainable agriculture. A large proportion of the phosphorus, nitrogen, sulfur, and transition metal elements that the host plant requires are obtained from the soil by the fungal mycelium and released at the arbuscules in exchange for photosynthates. While many of the plant transporters responsible for obtaining macronutrients at the periarbuscular space have been characterized, the identities of those mediating transition metal uptake remain unknown. In this work, MtCOPT2 has been identified as the only member of the copper transporter family COPT in the model legume Medicago truncatula to be specifically expressed in mycorrhizal roots. Fusing a C-terminal GFP tag to MtCOPT2 expressed under its own promoter showed a distribution pattern that corresponds with arbuscule distribution in the roots. When expressed in tobacco leaves, MtCOPT2-GFP co-localizes with a plasma membrane marker. MtCOPT2 is intimately related to the rhizobial nodule-specific MtCOPT1, which is suggestive of a shared evolutionary lineage that links transition metal nutrition in the two main root endosymbioses in legumes.

P0072GENERATION OF A C. ELEGANS MODEL TO STUDY THE INTERALLELIC INTERACTIONS OF PODOCIN

Abstract Background and Aims NPHS2 is the most frequently mutated gene in steroid-resistant nephrotic syndrome. NPHS2 encodes podocin, a key component of the glomerular filtration barrier. Podocin is a 42 kDa integral membrane protein, which accumulates in lipid raft microdomains at the podocyte slit diaphragm and is known to form oligomers. The c.686G>A, p.R229Q is the most common non-silent variant of NPHS2. We formerly described that R229Q is pathogenic only when trans-associated to specific 3’ missense mutations on the other parental allele. These C-terminal podocin mutants exert a dominant negative effect on R229Q podocin and retain it in intracellular compartments. As such, R229Q is the first variant in human genetics with a mutation-dependent pathogenicity. Based on FRET analysis and structural modeling, we formerly showed that the dominant negative effect is mediated by an altered oligomerization. However, the pathogenicity of several [mutation];[R229Q] associations is still in question and the identification of other rare NPHS2 variants with a mutation-dependent pathogenicity require an in vivo model. We therefore aimed to generate a Caenorhabditis elegans model, deficient for the homologous gene of NPHS2 (mec-2) and coexpressing differently (GFP- and mCherry-) tagged human podocin pairs. MEC-2 shares 45% identity and 83% similarity over 275aa (72%) of podocin (383aa). Expressed in six neurons, it is responsible for gentle-touch mechanosensation. The mec-2 mutants are insensitive to gentle touch in the center part of the body. Method Vectors encoding C-terminal GFP- or mCherry-tagged MEC-2 or human podocin with C. elegans codon optimization under mec-2 promoter and a selection marker (unc-119) were generated. Double (mec-2 and unc-119) mutant strains were established. Mutant strains were transformed by microparticle bombardment. The gentle-touch mechanosensation was examined by cat’s whiskers in a blinded experiment. Results The expression pattern of GFP and mCherry under mec-2 promoter corresponded to the six neurons responsible for gentle-touch sensation, indicating the proper functioning of the promoter. Strains with extrachromosomal MEC-2 or podocin coding vectors were successfully established (GFP-tagged MEC-2: n= 55 strains, GFP-tagged podocin: n= 81, mCherry-tagged podocin: n= 18). However, we found no rescue of the gentle touch sensation in any of them in blinded experiments. We hypothesized that either the fluorescent tag or the lack of chromosomal integration prevents the rescue effect of MEC-2. We therefore aimed to achieve self-cleaving of MEC-2 and the fluorescent tag, and inserted a T2A self-cleaving peptide-encoding sequence between them. To achieve chromosomal integration, we are implementing the MosSCI (Mos1-mediated Single Copy Insertion) technique. Conclusion Once the rescue with MEC-2 is achieved, the rescue effect of wild type and next different human podocin variant(s) will be aimed to analyze. The generation of the first animal model to study human interallelic interactions is challenging.

In-cell structural dynamics of an EGF receptor during ligand-induced dimer-oligomer transition.

The epidermal growth factor receptor (EGFR) is a membrane protein that regulates cell proliferation, differentiation and survival, and is a drug target for cancer therapy. Ligand-induced activation of the EGFR kinase is generally regarded to require ligand-bound-dimers, while phosphorylation and down-stream signalling is modulated by oligomers. Recent work has unveiled changes in EGFR dynamics from ligand-induced dimerization in membranes extracted from cells, however, less is known about the changes in EGFR dynamics that accompany the ligand-induced oligomerization in a live cell environment. Here, we determine the dynamics of a c-terminal GFP tag attached to EGFR in the unliganded dimer and in the liganded oligomers. By means of the single-frequency polarized phasor ellipse approach we extracted two correlation times on the sub-nanosecond and super-nanosecond timescales, respectively. EGF binding to the EGFR-GFP dimer lengthened the sub-nanosecond correlation time (from 0.1 to 1.3ns) and shortened the super-nanosecond correlation time (from 210 to 56ns) of the c-terminal GFP probe. The sub-nanosecond depolarization processes were assigned to electronic energy migration between proximal GFPs in the EGFR dimer or oligomer, while the super-nanosecond correlation times were assigned to nanosecond fluctuations of the GFP probe in the EGFR complex. Accordingly, these results show that ligand binding increased the average separation between the c-terminal tags and increased their rotational mobility. We propose that the dynamics are linked to an inhibitory function of the c-terminal tail in the un-liganded dimer and to the requirement of facile stochastic switching between kinase activation and cytoplasmic adaptor/effector binding in the active oligomers.

Strategies for successful isolation of a eukaryotic transporter

The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643–672 (OsBOR3Δ1-642). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-β-d-maltoside (DDM). The OsBOR3Δ1-642 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3Δ1-642 than DDM. Isolation of OsBOR3Δ1-642 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3Δ1-642 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3Δ1-642 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles.

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) Tcell therapy. In CAR Tcell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR Tcell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR Tcell therapy with properties, including rapid invivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR Tcell vectors stemming from this work are under clinical investigation.

Ectopic Expression of Rice PYL3 Enhances Cold and Drought Tolerance in Arabidopsis thaliana.

Abscisic acid (ABA) plays an important role in plant development and adaptation to abiotic stresses. The pyrabactin resistance-like (PYL) gene family has been characterized as intracellular ABA receptors in Arabidopsis. We describe here the functional characterization of PYL3 ABA receptor from a drought-tolerant rice landrace Nagina 22 (N22). The induced expression level of the PYL3 transcript was observed in the N22 under different stress treatments, including cold, drought, high temperature, salt and ABA. In contrast, the expression of PYL3 was down-regulated in drought-susceptible rice cv. IR64 in response to above stresses. C-terminal GFP translational fusion of OsPYL3 was localized to both cytosol and nucleus explaining in part functional conservation of PYL protein as ABA receptor. Arabidopsis transgenic lines overexpressing OsPYL3 were hypersensitive to ABA suggesting ABA signaling pathway-dependent molecular response of the OsPYL3. Further, constitutive overexpression of OsPYL3 in Arabidopsis led to improved cold and drought stress tolerance. Thus, OsPYL3 identified in this study could be a good candidate for genetic improvement of cold and drought stress tolerance of rice and other crop plants.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal.

AcCATPO is a plant catalase-phenol oxidase recently identified from red amaranth. Its physiological function remains unexplored. As the starting step of functional analysis, here we report its subcellular localization and a non-canonical targeting signal. Commonly used bioinformatics programs predicted a peroxisomal localization for AcCATPO, but failed in identification of canonical peroxisomal targeting signals (PTS). The C-terminal GFP tagging led the fusion protein AcCATPO-GFP to the cytosol and the nucleus, but N-terminal tagging directed the GFP-AcCATPO to peroxisomes and nuclei, in transgenic tobacco. Deleting the tripeptide (PTM) at the extreme C-terminus almost ruled out the peroxisomal localization of GFP-AcCATPOΔ3, and removing the C-terminal decapeptide completely excluded peroxisomes as the residence of GFP-AcCATPOΔ10. Furthermore, this decapeptide as a targeting signal could import GFP-10aa to the peroxisome exclusively. Taken together, these results demonstrate that AcCATPO is localized to the peroxisome and the nucleus, and its peroxisomal localization is attributed to a non-canonical PTS1, the C-terminal decapeptide which contains an internal SRL motif and a conserved tripeptide P-S/T-I/M at the extreme of C-terminus. This work may further the study as to the physiological function of AcCATPO, especially clarify its involvement in betalain biosynthesis, and provide a clue to elucidate more non-canonic PTS.

Cytosolic and Nuclear Co-localization of Betalain Biosynthetic Enzymes in Tobacco Suggests that Betalains Are Synthesized in the Cytoplasm and/or Nucleus of Betalainic Plant Cells.

Betalains replace anthocyanins as color pigments in most families of Caryophyllales. Unlike anthocyanins, betalains are derived from tyrosine via three enzymatic steps: hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA; step 1), and conversion of L-DOPA to betalamic acid (step 2), and to cyclo-DOPA (cDOPA; step 3). The principal enzymes responsible for these reactions have been elucidated at the molecular level, but their subcellular localizations have not been explored; hence, the intracellular compartments wherein betalains are biosynthesized remain unknown. Here, we report on the subcellular localization of these principal enzymes. Bioinformatic predictors and N- and C-terminal GFP tagging in transgenic tobacco, showed that Beta vulgaris CYP76AD1 which mediates both steps 1 and 3, DODA1 that catalyzes step 2, and CYP76AD6 which also mediates step 1, were similarly localized to the cytoplasm and nucleus (although the P450s were also weakly present in the endoplasmic reticulum). These two compartments were also the principal locations of Mirabilis jalapa cDOPA5GT. The cytoplasmic and nuclear co-localization of these key enzymes in tobacco suggests that betalains are biosynthesized in the cytoplasm and/or nucleus of betalain-containing plant cells. Elucidation of the subcellular compartmentation of betalain biosynthesis will facilitate the bioengineering of the betalain biosynthetic pathway in non-betalain-containing plants.