Endoplasmic Reticulum Retention Signal Research Articles

Characterizing interactions of endoplasmic reticulum resident proteins in situ through the YST-PPI method.

The mutual interactions of endoplasmic reticulum (ER) resident proteins in the ER maintain its functions, prompting the protein folding, modification, and transportation. Here, a new method, named YST-PPI (YESS-based Split fast TEV protease system for Protein-Protein Interaction) was developed, targeting the characterization of protein interactions in ER. YST-PPI method integrated the YESS system, split-TEV technology, and endoplasmic reticulum retention signal peptide (ERS) to provide an effective strategy for studying ER in situ PPIs in a fast and quantitative manner. The interactions among 15 ER-resident proteins, most being identified molecular chaperones, of S. cerevisiae were explored using the YST-PPI system, and their interaction network map was constructed, in which more than 74 interacting resident protein pairs were identified. Our studies also showed that Lhs1p plays a critical role in regulating the interactions of most of the ER-resident proteins, except the Sil1p, indicating its potential role in controlling the ER molecular chaperones. Moreover, the mutual interaction revealed by our studies further confirmed that the ER-resident proteins perform their functions in a cooperative way and a multimer complex might be formed during the process.

The role and mechanism of TXNDC5 in disease progression.

Thioredoxin domain containing protein-5 (TXNDC5), also known as endothelial protein-disulfide isomerase (Endo-PDI), is confined to the endoplasmic reticulum through the structural endoplasmic reticulum retention signal (KDEL), is a member of the PDI protein family and is highly expressed in the hypoxic state. TXNDC5 can regulate the rate of disulfide bond formation, isomerization and degradation of target proteins through its function as a protein disulfide isomerase (PDI), thereby altering protein conformation, activity and improving protein stability. Several studies have shown that there is a significant correlation between TXNDC5 gene polymorphisms and genetic susceptibility to inflammatory diseases such as rheumatoid, fibrosis and tumors. In this paper, we detail the expression characteristics of TXNDC5 in a variety of diseases, summarize the mechanisms by which TXNDC5 promotes malignant disease progression, and summarize potential therapeutic strategies to target TXNDC5 for disease treatment.

Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms.

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Transcription factor NAC1 activates expression of peptidase-encoding AtCEPs in roots to limit root hair growth.

Plant genomes encode a unique group of papain-type Cysteine EndoPeptidases (CysEPs) containing a KDEL endoplasmic reticulum (ER) retention signal (KDEL-CysEPs or CEPs). CEPs process the cell-wall scaffolding EXTENSIN (EXT) proteins that regulate de novo cell-wall formation and cell expansion. Since CEPs cleave EXTs and EXT-related proteins, acting as cell-wall-weakening agents, they may play a role in cell elongation. The Arabidopsis (Arabidopsis thaliana) genome encodes 3 CEPs (AtCPE1-AtCEP3). Here, we report that the genes encoding these 3 Arabidopsis CEPs are highly expressed in root-hair (RH) cell files. Single mutants have no evident abnormal RH phenotype, but atcep1-3 atcep3-2 and atcep1-3 atcep2-2 double mutants have longer RHs than wild-type (Wt) plants, suggesting that expression of AtCEPs in root trichoblasts restrains polar elongation of the RH. We provide evidence that the transcription factor NAC1 (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) activates AtCEPs expression in roots to limit RH growth. Chromatin immunoprecipitation indicates that NAC1 binds to the promoter of AtCEP1, AtCEP2, and, to a lower extent, AtCEP3 and may directly regulate their expression. Inducible NAC1 overexpression increases AtCEP1 and AtCEP2 transcript levels in roots and leads to reduced RH growth while the loss of function nac1-2 mutation reduces AtCEP1-AtCEP3 gene expression and enhances RH growth. Likewise, expression of a dominant chimeric NAC1-SRDX repressor construct leads to increased RH length. Finally, we show that RH cell walls in the atcep1-3 atcep3-2 double mutant have reduced levels of EXT deposition, suggesting that the defects in RH elongation are linked to alterations in EXT processing and accumulation. Our results support the involvement of AtCEPs in controlling RH polar growth through EXT processing and insolubilization at the cell wall.

The intracellular C-terminal domain of mGluR6 contains ER retention motifs

Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

Abstract 690: Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses

Abstract Many cancer immunotherapies require the strong activation of the T-cell mediated adaptive immune response to elicit their full anti-tumor potential. Antigen processing and presentation is a complex, multistep process that can be significantly enhanced via antigen modification, leading to better antigen-specific immune responses. Using mRNA, we can express exogenous antigens inside the cell and facilitate the subcellular localization of these antigens to enhance antigen processing and presentation. In this work, we introduced endolysosomal trafficking domains from CD1d molecules to drive subcellular localization of antigenic proteins to the late endosomes (LE) to enhance class I and class II antigen loading. CD1d are molecules that naturally traffic between the membrane and the late endosomes, where antigens are processed and loaded to both class II and class I molecules in a TAP-independent manner. We compared the immunogenicity of a novel endolysosomal trafficking domain, human CD1d, coupled to a chimeric HPV16 E6 and E7 (HPV16E6E7-hCD1d) antigen to antigen designs intended to be either a) secreted (Sec-HPV16E6E7), b) retained in the endoplasmic reticulum (ER) by an ER retention signal KDEL (HPV16E6E7-KDEL) or c) unmodified antigen sequence (HPV16E6E7). To ensure intracellular translation and expression, all antigens were encoded and delivered as mRNAs. The immunogenicity of each antigen design was assessed in HPV16 positive cervical intraepithelial neoplasia (CIN) donor derived PBMCs and healthy controls. Using confocal microscopy, we observed that the hCD1d trafficking domain directed the translated E6E7 protein into late endosomes, which was not observed for any of the other antigen designs. Importantly, antigen localization to late endosomes resulted in significantly improved immunogenicity in vitro as well as in vivo. We observed a consistent increase in numbers of activated antigen-specific T cells across multiple CIN donor PBMCs treated with HPV16E6E7-hCD1d compared to the other 3 designs, as measured by Interferon gamma secretion, surface marker expression, and T-cell proliferation. To assess the generalizability of this approach, we also compared immunogenicity in human donor PBMCs using the human cytomegalovirus antigen pp65, and observed similar results. Lastly, the immunogenicity of each antigen design was tested in vivo by intramuscular injection of nanoparticle formulated mRNAs in C56/Bl6 mice, and inclusion of the murine analogue (mCD1d) resulted in significantly increased generation of HPV16-specific T cell responses. Based on these findings, we are developing a novel mRNA-based cancer therapeutic that includes the addition of hCD1d trafficking domain in the HPV16-specific antigen component. Citation Format: Weiqun Liu, Daniel Fernandez, Colin J. McKinlay, Daniel O. Frimannsson, Meredith L. Leong, W. Martin Kast, Samuel Deutsch, Ole Audun W. Haabeth. Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 690.

A novel fluorescent endoplasmic reticulum marker for super-resolution imaging in live cells.

Endoplasmic reticulum (ER) is a highly complicated and dynamic organelle that actively changes its shape and communicates with other organelles. Visualization of ER in live cells is of great importance to understand cellular activities. Here, we designed a novel ER marker, RR-mNeonGreen, which comprised an N-terminal ER retention signal, a bright fluorescent protein (mNeonGreen), and a C-terminal transmembrane region. Colocalization of RR-mNeonGreen with mCherry-KDEL verified that RR-mNeonGreen perfectly labeled the ER. RR-mNeonGreen showed better continuity of ER tubules when imaged by super-resolution microscopy. Moreover, RR-mNeonGreen is competent for live-cell imaging of ER dynamics and tracing of the interaction between ER and mitochondria at high spatiotemporal resolution. In summary, RR-mNeonGreen is a novel ER marker for super-resolution live-cell imaging with multiple merits.

Endoplasmic Reticulum Bodies in the Lateral Root Cap Are Involved in the Direct Transport of Beta-Glucosidase to Vacuoles.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the β-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport β-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.

Mutant MESD links cellular stress to type I collagen aggregation in osteogenesis imperfecta type XX

Aberrant forms of endoplasmic reticulum (ER)-resident chaperones are implicated in loss of protein quality control in rare diseases. Here we report a novel mutation (p.Asp233Asn) in the ER retention signal of MESD by whole exome sequencing of an individual diagnosed with osteogenesis imperfecta (OI) type XX. While MESDD233N has similar stability and chaperone activity as wild-type MESD, its mislocalization to cytoplasm leads to imbalance of ER proteostasis, resulting in improper folding and aggregation of proteins, including LRP5 and type I collagen. Aggregated LRP5 loses its plasma membrane localization to disrupt the expression of WNT-responsive genes, such as BMP2, BMP4, in proband fibroblasts. We show that MESD is a direct chaperone of pro-α1(I) [COL1A1], and absence of MESDD233N in ER results in cytosolic type I collagen aggregates that remain mostly not secreted. While cytosolic type I collagen aggregates block the intercellular nanotubes, decreased extracellular type I collagen also results in loss of interaction of ITGB1 with type I collagen and weaker attachment of fibroblasts to matrix. Although proband fibroblasts show increased autophagy to degrade the aggregated type I collagen, an overall cellular stress overwhelms the proband fibroblasts. In summary, we present an essential chaperone function of MESD for LRP5 and type I collagen and demonstrating how the D233N mutation in MESD correlates with impaired WNT signaling and proteostasis in OI.

A splicing-dependent ER retention signal regulates surface expression of the mechanosensitive TMEM63B cation channel

TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon 4 in TMEM63B. The short variant lacking exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.

Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice.

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

The Humanised NPY-mRFP RBL Reporter Cell Line Is a Fast and Inexpensive Tool for Detection of Allergen-Specific IgE in Human Sera.

Rat basophilic leukaemia (RBL) cells have been used for decades as a model of high-affinity Immunoglobulin E (IgE) receptor (FcεRI) signalling. Here, we describe the generation and use of huNPY-mRFP, a new humanised fluorescent IgE reporter cell line. Fusion of Neuropeptide Y (NPY) with monomeric red fluorescent protein (mRFP) results in targeting of fluorescence to the granules and its fast release into the supernatant upon IgE-dependent stimulation. Following overnight sensitisation with serum, optimal release of fluorescence upon dose-dependent stimulation with allergen-containing extracts could be measured after 45 min, without cell lysis or addition of any reagents. Five substitutions (D194A, K212A, K216A, K226A, and K230A) were introduced into the FcεRIα cDNA used for transfection, which resulted in the removal of known endoplasmic reticulum retention signals and high surface expression of human FcεRIα* in huNPY-mRFP cells (where * denotes the penta-substituted variant), comparable to the ~500,000 FcεRIα molecules per cell in the RS-ATL8 humanised luciferase reporter, which is a human FcεRIα/FcεRIγ double transfectant. The huNPY-mRFP reporter was used to demonstrate engagement of specific IgE in sera of Echinococcus granulosus-infected individuals by E. granulosus elongation factor EgEF-1β and, to a lesser extent, by EgEF-1δ, which had been previously described as IgE-immunoreactive EgEF-1β/δ.

Dominant-Negative Form of SIGIRR: SIGIRRΔE8 Promotes Tumor Growth Through Regulation of Metabolic Pathways

Colorectal carcinoma is the leading cause of cancer-related death. Previously we have shown that tumor suppressor single immunoglobulin interleukin-1-related receptor (SIGIRR) is frequently inactivated in human colorectal cancer by the increased expression of a novel SIGIRR isoform (SIGIRRΔE8). SIGIRRΔE8 showed increased retention in the cytoplasm and loss of complex glycan modification compared to the full-length SIGIRR. Now we found that the arginine residues located in the C-terminus of SIGIRRΔE8 serve as an endoplasmic reticulum retention signal and are required for resident protein ribophorin 1 (RPN1) interaction. In addition, we found that SIGIRRΔE8 exerts a direct impact on cell metabolism through interaction with the adenosine triphosphate synthase in the colorectal cancer cells. SIGIRRΔE8 expression promoted the metabolic shift through upregulation of mammalian target of rapamycin signaling pathway and dysregulation of mitochondrial function to promote survival and proliferation of colon cancer cells in xenograft model.

Enhancing GluN2A-type NMDA receptors impairs long-term synaptic plasticity and learning and memory.

N-methyl-D-aspartic acid type glutamate receptors (NMDARs) play critical roles in synaptic transmission and plasticity, the dysregulation of which leads to cognitive defects. Here, we identified a rare variant in the NMDAR subunit GluN2A (K879R) in a patient with intellectual disability. The K879R mutation enhanced receptor expression on the cell surface by disrupting a KKK motif that we demonstrated to be an endoplasmic reticulum retention signal. Expression of GluN2A_K879R in mouse hippocampal CA1 neurons enhanced the excitatory postsynaptic currents mediated by GluN2A-NMDAR but suppressed those mediated by GluN2B-NMDAR and the AMPA receptor. GluN2A_K879R knock-in mice showed similar defects in synaptic transmission and exhibited impaired learning and memory. Furthermore, both LTP and LTD were severely impaired in the KI mice, likely explaining their learning and memory defects. Therefore, our study reveals a new mechanism by which elevated synaptic GluN2A-NMDAR impairs long-term synaptic plasticity as well as learning and memory.

Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Response of protein secretion based on transcriptional control is rather slow, as it requires transcription, translation and transport from the endoplasmic reticulum (ER) to the plasma membrane via the conventional protein secretion (CPS) pathway. An alternative regulation to provide faster response would be valuable. Here we present two genetically encoded orthogonal regulatory secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by a cytosolic protease) or inside the ER lumen (lumER, released by an ER-luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables the combination of signals into logic functions. Its application was demonstrated on a chemically regulated therapeutic protein secretion and regulated membrane translocation of a chimeric antigen receptor (CAR) targeting cancer antigen. Regulation of the ER escape represents a platform for the design of fast-responsive and tightly-controlled modular and scalable protein secretion system for mammalian cells.

Overexpression of PvFAD3 Gene from Plukenetia volubilis Promotes the Biosynthesis of α-Linolenic Acid in Transgenic Tobacco Seeds.

The ω-3 fatty acid desaturase (FAD3) gene encodes a rate-limiting enzyme in the synthesis of α-linolenic acid. In this study, homologous cloning was used to obtain the full-length sequence of the PvFAD3 gene of Plukenetia volubilis. The full-length DNA sequence was 1871 bp long, with 8 exons and 7 introns. The structural analysis of the amino acid sequence revealed that the PvFAD3 protein contained three histidine-conserved regions and an endoplasmic reticulum retention signal. The real-time reverse transcription-polymerase chain reaction performed for determining the expression patterns of the PvFAD3 gene in different tissues of P. volubilis showed that PvFAD3 expression was highly expressed in the fast oil accumulation stage of seed. The analysis of subcellular localization assay in epidermal cells of tobacco (Nicotiana benthamiana) leaves showed that the PvFAD3 protein was mainly localized in the endoplasmic reticulum. Seed-specific overexpression vectors were constructed, and Agrobacterium-mediated genetic transformation was performed to obtain transgenic tobacco plants overexpressing PvFAD3. The results of fatty acid assays performed using harvested seeds showed a significant increase in α-linolenic acid content, a dramatic decrease in linoleic acid content, and an obvious increase in oil content in transgenic tobacco seeds. Collectively, the PvFAD3 gene of P. volubilis was confirmed as a key enzyme gene for α-linolenic acid synthesis; thus, indicating that the PvFAD3 gene can be used for fatty acid fraction improvement in oilseed plants.

Targeting human CALR-mutated MPN progenitors with a neoepitope-directed monoclonal antibody.

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co‐expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK‐STAT signalling, TPO‐independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR‐dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.

Effect of an Endoplasmic Reticulum Retention Signal Tagged to Human Anti-Rabies mAb SO57 on Its Expression in Arabidopsis and Plant Growth.

Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium-mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T1 transformants and obtained homozygous T3 seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.