Endoplasmic Reticulum Retention Sequence Research Articles

AGR2: a secreted protein worthy of attention in diagnosis and treatment of breast cancer.

AGR2 is a secreted protein widely existing in breast. In precancerous lesions, primary tumors and metastatic tumors, the expression of AGR2 is increased, which has aroused our interest. This review introduces the gene and protein structure of AGR2. Its endoplasmic reticulum retention sequence, protein disulfide isomerase active site and multiple protein binding sequences endow AGR2 with diverse functions inside and outside breast cancer cells. This review also enumerates the role of AGR2 in the progress and prognosis of breast cancer, and emphasizes that AGR2 can be a promising biomarker and a target for immunotherapy of breast cancer, providing new ideas for early diagnosis and treatment of breast cancer.

Plant-derived PAP proteins fused to immunoglobulin A and M Fc domains induce anti-prostate cancer immune response in mice

In this study, recombinant Fc-fused Prostate acid phosphatase (PAP) proteins were produced in transgenic plants. PAP was fused to immunoglobulin (Ig) A and M Fc domain (PAP-IgA Fc and PAP-IgM Fc), which were tagged to the ER retention sequence KDEL to generate PAP-IgA FcK and PAP-IgM FcK. Agrobacterium-meditated transformation was performed to produce transgenic tobacco plants expressing four recombinant proteins. Genomic PCR and RT-PCR analyses confirmed the transgene insertion and mRNA transcription of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK in tobacco plant leaves. Western blot confirmed the expression of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK proteins. SEC-HPLC and Bio-TEM analyses were performed to confirm the size and shape of the plant-derived recombinant PAP-Fc fusion proteins. In mice experiments, the plant-derived IgA and IgM Fc fused proteins induced production of total IgGs including IgG1 against PAP. This result suggests that IgA and IgM Fc fusion can be applied to produce recombinant PAP proteins as a prostate cancer vaccine in plant expression system.

Formation of ER-lumenal intermediates during export of Plasmodium proteins containing transmembrane-like hydrophobic sequences.

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER-lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway.

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

Calreticulin – a multifaced protein

Summary Calreticulin (CALR) is a highly conserved multi-function protein that primarily localizes within the lumen of the endoplasmic reticulum (ER). It participates in various processes in the cells, including glycoprotein chaperoning, regulation of Ca2+ homeostasis, antigen processing and presentation for adaptive immune response, cell adhesion/migration, cell proliferation, immunogenic cell death, gene expression and RNA stability. The role of CALR in the assembly, retrieval and cell surface expression of MHC class I molecules is well known. A fraction of the total cellular CALR is localized in the cytosol, following its retro-translocation from the ER. In the cell stress conditions, CALR is also expressed on the cell surface via an interaction with phosphatidylserine localized on the inner leaflet of the plasma membrane. The abovementioned mechanism is relevant for the recognition of the cells, as well as immunogenicity and phagocytic uptake of proapoptotic and apoptotic cells. Lastly, the presence of CALR exon 9 gene mutations was confirmed in patients with myeloproliferative neoplasms. Their presence results in an abnormal CALR structure due to the loss of its ER-retention sequence, CALR extra-ER localisation, the formation of a complex with thrombopoietin receptor, and oncogenic transformation of hematopoietic stem cells. It is also known that CALR exon 9 mutants are highly immunogenic and induce T cell response. Despite this fact, CALR mutant positive hematopoietic cells emerge. The last phenomenon is probably the result of the inhibition of phagocytosis of the cancer cells exposing CALR mutant protein by dendritic cells.

RAP Tag and PMab-2 Antibody: A Tagging System for Detecting and Purifying Proteins in Plant Cells.

An affinity tag system requires both high affinity and specificity. The RAP tag epitope DMVNPGLEDRIE, derived from rat podoplanin (PDPN), is specifically recognized by PMab-2 monoclonal antibodies in rats. Here, we demonstrated that high levels of PMab-2 can be produced in Nicotiana benthamiana and plant-derived PMab-2 possesses similar activity to CHO-derived PMab-2, and the RAP tag presents a useful tagging system for detecting and purifying proteins from plant cells. The heavy chain of PMab-2 fused with KDEL, an endoplasmic reticulum retention sequence, and the light chain of the antibody were introduced into N. benthamiana by agroinfiltration. The expression of PMab-2 peaked 4 days after agroinfiltration, and approximately 0.3 mg/g fresh weight of the antibody was accumulated. After purification, the plant-derived PMab-2 successfully recognized rat PDPN expressed in CHO-K1 cells and exhibited almost the same binding activity as CHO-derived PMab-2. The RAP-tagged proteins expressed in plant cells were specifically recognized by PMab-2. These results indicate that PMab-2 can accumulate at high levels in N. benthamiana and is easily purified and that the RAP tagging system presents a useful tool for detecting and purifying proteins of interest in plant cells.

Trophic activities of endoplasmic reticulum proteins CDNF and MANF.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are endoplasmic reticulum (ER) luminal proteins that confer trophic activities in a wide range of tissues under diverse pathological conditions. Despite initially being classified as neurotrophic factors, neither protein structurally nor functionally resembles bona fide neurotrophic factors. Their highly homologous structures comprise a unique globular, saposin-like domain within the N-terminus joined by a flexible linker to a C-terminus containing a SAP-like domain, CXXC motif and an ER retention sequence. Neurotrophic factors exert effects by binding to cognate receptors in the plasma membrane; however, no cell surface receptors have been identified for MANF and CDNF. Both can act as unfolded protein response (UPR) genes that modulate the UPR and inflammatory processes. The trophic activity of MANF and CDNF extends beyond the central nervous system with MANF being crucial for the development of pancreatic β cells and both have trophic effects in a variety of diseases related to the liver, heart, skeletal tissue, kidney and peripheral nervous system. In this article, the unique features of MANF and CDNF, such as their structure and mechanisms of action related to ER stress and inflammation, will be reviewed. Recently identified interactions with lipids and membrane trafficking will also be described. Lastly, their function and therapeutic potential in different diseases including a recent clinical trial using CDNF to treat Parkinson's disease will be discussed. Collectively, this review will highlight MANF and CDNF as broad-acting trophic factors that regulate functions of the endoplasmic reticulum.

Solution structure of human myeloid-derived growth factor suggests a conserved function in the endoplasmic reticulum

Human myeloid-derived growth factor (hMYDGF) is a 142-residue protein with a C-terminal endoplasmic reticulum (ER) retention sequence (ERS). Extracellular MYDGF mediates cardiac repair in mice after anoxic injury. Although homologs of hMYDGF are found in eukaryotes as distant as protozoans, its structure and function are unknown. Here we present the NMR solution structure of hMYDGF, which consists of a short α-helix and ten β-strands distributed in three β-sheets. Conserved residues map to the unstructured ERS, loops on the face opposite the ERS, and the surface of a cavity underneath the conserved loops. The only protein or portion of a protein known to have a similar fold is the base domain of VNN1. We suggest, in analogy to the tethering of the VNN1 nitrilase domain to the plasma membrane via its base domain, that MYDGF complexed to the KDEL receptor binds cargo via its conserved residues for transport to the ER.

Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression.

For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries.

Dysregulated IL-6/GP130/JAK Signaling in Calreticulin Mutated Myeloproliferative Neoplasms (MPN)

Introduction. Frameshift mutations in exon 9 of calreticulin (CALR) gene represent the second most frequent alteration in MPN. More than 30 types of CALR mutations are described; the most frequent are a 52bp deletion (Type1, T1) and a 5bp insertion (T2). All mutations cause a recurrent frameshift that originates a novel C-terminal peptide lacking the KDEL Endoplasmic Reticulum-retention sequence. Recent data described the interaction of mutated CALR (CALRm) with the thrombopoietin receptor MPL and the resulting activation of JAK/STATs as well MAPK and PI3K pathways (Araki M et al & Chachoua I et al, Blood 2016; Elf S et al, Blood 2018). However, full characterization of the molecular mechanisms linking CALRm with MPN remains to be fully accomplished. Methods. By using CRISPR/Cas9 technology we generated CALR knock out (KO) and T1 variants from the GM-CSF cytokine dependent UT7 cell line; primary CD34+ cells from CALRm pts were also analyzed. Results. Introduction of CALR T1 mutation or generation of KO status did not appreciably modified the proliferation rate, cell cycle and apoptosis of UT7 cells under standard conditions. However, UT7 T1 and KO cells were similarly able to grow in the absence of GM-CSF, otherwise necessary for WT cells; in these cytokine-depleted conditions, apoptosis was significantly increased in WT cells compared both T1 and KO (4- and 8-fold higher; p<0.01) suggesting acquisition of cytokine independency. Exploring the mechanisms leading to cytokine independence we found that signal transducer and activation of transcription 3 (STAT3) was the main constitutively phosphorylated STAT in T1 and KO cells. Among a panel of STAT3 activation candidates assessed by RT-PCR, we found that mRNA levels of interleukin 6 (IL6), which induces STAT3 phosphorylation and activation through the glycoprotein 130/Janus kinase (GP130/JAK) pathway, were significantly increased in T1 and KO compared to parental cells (2- and 3.5-fold higher; p<0.01). The levels of IL6 released in cell culture medium were also significantly increased (1.5- and 2-fold higher; p<0.05). Blocking GP130 with SC144 or IL6 receptor (IL6R) with Tocilizumab (TCZ), in cytokine-depleted conditions, led to cell growth arrest in T1 and KO with no effects on WT cells. SC144 treatment induced a marked dephosphorylation of STAT3 in T1 and KO cells. Also, the membrane expression of GP130 and IL6R resulted higher in KO and T1 compared to WT cells, whilst intracellular content was similar. Co-immunoprecipitation showed that wild type calreticulin, unlike the mutated T1, formed complexes with GP130 and IL6R suggesting that CALRm lost the capacity to negatively regulate their membrane exposure resulting in downstream increased IL6 synthesis and release. The role of IL6 pathway in CALRm patients was evaluated by assessing megakaryocyte (Mk) colony generation in plasma clot cultures of CD34+ cells in the absence/presence of IL6, as part of cytokine cocktail optimally supporting CFU-Mk growth. We found that CFU-Mk generation by CALRm CD34+ cells was largely independent of exogenously added IL6, unlike control CFU-MK whose number was halved in the absence of IL6 (p<0.05). Moreover, inhibition of GP130 and IL6R by SC144 and TCZ, respectively, significantly reduced CFU-Mk by CALRm CD34+ cells (2.9- and 3.2-fold; p<0.05), with no effect in control cultures. As anticipated, JAK inhibitors ruxolitinib and momelotinib reduced CFU-Mk formation by CALRm CD34+ cells (2- and 3.2-fold, respectively; p<0.01), particularly evident in exogenous IL6-free conditions (5.8- and 4.8-fold, respectively; p<0.01). The combination of either ruxolitinib or momelotinib with TCZ on CFU-Mk formation was also assessed. Both combinations proved synergistic activity in reducing CFU-Mk from CALRm CD34+ cells (4.3- and 5-fold vs control cultures, respectively; p<0.01; combination compared to single agent, 2.8- fold vs ruxolitinib, 4.5-fold vs momelotinib and 4-fold vs TCZ, p<0.05 for all). Conclusions. Overall, our data suggest a novel model of action of mutant CALR resulting from the loss of its capability to complex GP130 and IL6R and regulate their cell membrane expression. Augmented expression of IL6 receptor complex on CALRm CD34+ cells on turn results in deregulated IL6-dependent signaling and augmented megakaryocyte generation. Blockade of IL-6/GP130/JAK signaling pathway might represent a new therapeutic target in pts with CALR mutation. Disclosures Vannucchi: Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

In vitro leishmanicidal activity of antimicrobial peptide KDEL against Leishmania tarentolae.

Leishmaniasis, caused by the intracellular protozoan parasite Leishmania, remains an important neglected tropical infectious disease. Infection may be lethal if untreated. Currently, the available drugs for the disease are limited by high toxicity and drug resistance. There is an urgent need to develop novel anti-leishmanial strategies. Antimicrobial peptides (AMPs) have been described as the first-line immune defense against pathogenic microbes and are being developed as emerging anti-parasitic therapies. In the present study, we showed the anti-leishmanial activity of the synthetic 4-amino acid peptide lysine, aspartic acid, glutamic acid, and leucine (KDEL), the endoplasmic reticulum retention sequence, against Leishmania tarentolae promastigote and amastigote. Different concentrations of KDEL peptides were incubated with promastigotes, MTT viability assay, and promastigote assay were carried out. Macrophages infected with GFP-transfected L. tarentolae promastigotes were incubated with KDEL peptides, and the anti-amastigote activity of the KDEL peptides was measured by fluorescence microscopy. The damage of L. tarentolae was observed by light microscopy and electron microscopy. The cell apoptosis was analyzed using the Annexin V-FITC/PI apoptosis detection kit and mitochondrial membrane potential assay kit and by flow cytometry. Results showed that L. tarentolae was susceptible to KDEL peptides in a dose-dependent manner, and KDEL peptides disrupted the surface membrane integrity and caused cell apoptosis. In our study, we found for the first time an AMP KDEL from Pseudomonas aeruginosa and proved its significant therapeutic potential as a novel anti-leishmanial drug.

KDEL Receptor 1 Contributes to Cell Surface Association of Protein Disulfide Isomerases

Endoplasmic reticulum (ER)-resident proteins with a C-terminal KDEL ERretention sequence are captured in the Golgi apparatus by KDEL receptors (KDELRs). The binding of such proteins to these receptors induces their retrograde transport. Nevertheless, some KDEL proteins, such as Protein Disulfide Isomerases (PDIs), are found at the cell surface. PDIs target disulfide bridges in the extracellular domains of proteins, such as integrins or A Disintegrin And Metalloprotease 17 (ADAM17) leading to changes in the structure and function of these molecules. Integrins become activated and ADAM17 inactivated upon disulfide isomerization. The way that PDIs escape from retrograde transport and reach the plasma membrane remains far from clear. Various mechanisms might exist, depending on whether a local cell surface association or a more global secretion is required. To get a more detailed insight in the transport of PDIs to the cell surface, methods such as cell surface biotinylation, flow cytometric analysis, immunoprecipitation, fluorescence microscopy as well as labeling of cells with fluorescence labled recombinant PDIA6 was performed. Here, we show that the C-terminal KDEL ER retention sequence is sufficient to prevent secretion of PDIA6 into the extracellular space but is mandatory for its association with the cell surface. The cell surface trafficking of PDIA1, PDIA3, and PDIA6 is dependent on KDELR1, which travels in a dynamic manner to the cell surface. This transport is assumed to result in PDI cell surface association, which differs from PDI inducible secretion into the extracellular space. Distinct PDIs differ in their trafficking properties. Endogenous KDELR1, detectable at the cell surface, might be involved not only in the transport of cell-surface-associated PDIs, but also in their retrieval and internalization from the extracellular space. Beside their ER retention motive PDIs travel to the cell surface. Here they target different proteins to render their function. To escape the ER PDIs travel via various pathways. One of them depends on the KDELR1, which can transport its target to the cell surface, where it is to be expected to release its cargo in close vicinity to its target molecules. Hence, the KDEL sequence is needed for cell surface association of PDIs, such as PDIA6.

KDEL Receptors Are Differentially Regulated to Maintain the ER Proteome under Calcium Deficiency

Retention of critical endoplasmic reticulum (ER) luminal proteins needed to carry out diverse functions (e.g., protein synthesis and folding, lipid metabolism) is mediated through a carboxy-terminal ER retention sequence (ERS) and its interaction with KDEL receptors. Here, we demonstrate that depleting ER calcium causes mass departure of ERS-containing proteins from cells by overwhelming KDEL receptors. In addition, we provide evidence that KDELR2 and KDELR3, but not KDELR1, are unfolded protein response (UPR) genes upregulated as an adaptive response to counteract the loss of ERS-containing proteins, suggesting previously unknown isoform-specific functions of the KDEL receptors. Overall, our findings establish that decreases in ER calcium change the composition of the ER luminal proteome and secretome, which can impact cellular functions and cell viability. The redistribution of the ER proteome from inside the cell to the outside has implications for dissecting the complex relationship of ER homeostasis with diverse disease pathologies.

CDNF induces the adaptive unfolded protein response and attenuates endoplasmic reticulum stress-induced cell death

The Cerebral Dopamine Neurotrophic Factor (CDNF) is a neurotrophic factor that has a protective effect in cell and animal models of several neurodegenerative diseases. The molecular mechanism of the protective effect of CDNF is unclear. Many neurodegenerative diseases have been related to a proteostasis dysregulation in the endoplasmic reticulum (ER). A failure of proteostasis produces ER stress, triggering the unfolded protein response (UPR) and, in the long-term, induces cell death. An adaptive UPR solves ER stress by attenuating protein synthesis, inducing chaperones expression, and degradation of misfolded proteins. Since CDNF is an ER resident protein, we investigated whether the role of CDNF is to regulate ER proteostasis. To this end, we determined the effect of CDNF in thapsigargin-induced ER stress in HEK293-T cells and cultured hippocampal neurons. Our results show that CDNF improved the viability of HEK293-T cells exposed to thapsigargin. CDNF increased levels of protective proteins of the early UPR, such as BiP, ATF4, ATF6, and XBP-1 in both HEK293-T cells and neurons. Conversely, expression of CDNF attenuated ER stress-induced apoptotic proteins, CHOP and cleaved caspase-3 in HEK293-T cells and neurons. A mutant CDNF lacking the ER retention sequence failed to protect against ER stress. In conclusion, CDNF regulates proteostasis in the ER by inducing the adaptive UPR response and inhibiting apoptotic pathways triggered by ER stress. We propose that neuroprotection induced by CDNF is mediated by regulating ER proteostasis.

Tumor-associated calreticulin variants functionally compromise the peptide loading complex and impair its recruitment of MHC-I

Major histocompatibility complex-I-β2m dimers (MHC-I) bind peptides derived from intracellular proteins, enabling the immune system to distinguish between normal cells and those expressing pathogen-derived or mutant proteins. The peptides bind to MHC-I in the endoplasmic reticulum (ER), and this binding is facilitated by the peptide loading complex (PLC), which contains calreticulin (CRT). CRT associates with MHC-I via a conserved glycan present on MHC-I and recruits it to the PLC for peptide binding. Somatic frameshift mutations in CRT (CRT-FS) drive the proliferation of a subset of myeloproliferative neoplasms, which are chronic blood tumors. All CRT-FS proteins have a C-terminal sequence lacking the normal ER-retention signal and possessing a net negative charge rather than the normal positive charge. We characterized the effect of CRT-FS on antigen presentation by MHC-I in human cells. Our results indicate that CRT-FS cannot mediate CRT's peptide loading function in the PLC. Cells lacking CRT exhibited reduced surface MHC-I levels, consistent with reduced binding of high-affinity peptides, and this was not reversed by CRT-FS expression. CRT-FS was secreted and not detectably associated with the PLC, leading to poor MHC-I recruitment, although CRT-FS could still associate with MHC-I in a glycan-dependent manner. The addition of an ER-retention sequence to CRT-FS restored its association with the PLC but did not rescue MHC-I recruitment or its surface expression, indicating that the CRT-FS mutants functionally compromise the PLC. MHC-I down-regulation permits tumor cells to evade immune surveillance, and these findings may therefore be relevant for designing effective immunotherapies for managing myeloproliferative neoplasms.

Antitumor Response by Endoplasmic Reticulum-Targeting DNA Vaccine Is Improved by Adding a KDEL Retention Signal.

Directing an antigen to the endoplasmic reticulum (ER) improves the antigen-specific immune response, revealing a potentially useful strategy in cancer immunotherapy using tumor-associated antigens (TAAs). This can be achieved by fusing the antigen to an ER chaperone protein, such as calreticulin (CRT). We previously reported the antitumor response by fusing the CRT signal peptide (SP) and its ER retention sequence (KDEL) to full-length human papillomavirus type 16 (HPV-16) E6 and E7 antigens, obtaining a potent antitumoral effect. In this article, we compare the antitumor response due to the use of each signal (SP and/or KDEL) fused to HPV16 E6 and E7 antigens in a DNA vaccination model. Using both SP and KDEL signals promotes higher interferon (IFN)-γ production and a faster antitumor response than using only the SP, resulting in better tumor growth restraint and higher survival, indicating that the KDEL addition to an ER-directed antigen helps by shortening the time to response. Meanwhile, antigens without signals or only the KDEL signal showed no induction of antigen-specific IFN-γ or antitumor response. Our results indicate that directing the E6E7m antigen to the ER by the SP signal is sufficient to promote an efficient antitumor response. Importantly, this effect is stronger and faster when the antigen also has an ER retention sequence, such as the KDEL signal.

The Role of the Furin‐Cleavable Linker and KDEL Sequence in Cytotoxicity of Recombinant ImmunoDNase

The lack of specificity of standard chemotherapies to identify and kill cancer cells has challenged researchers to engineer targeted therapies to replace conventional treatments. One strategy that is currently being optimized to treat cancer is the use of recombinant immunotoxins (RITs). RITs are chimeric proteins that are engineered to contain an antibody conjugated to a toxin. The antibody allows for specific targeting of cancer cells while the toxin domain is internalized by the targeted cell and subsequently induces cell death. These therapies suffer from several problems, including non‐specific toxicities, low anti‐tumor activity, and immunogenicity. A bacterial moiety introduced into a patient with an intact immune system can elicit an immune response that consequently eliminates the possibility of retreatment. Deimmunized, humanized, and fully human immunoconjugates have been engineered, but often lack efficacy. We are using specific sequences known to assist cellular intoxication by Pseudomonas exotoxin A (PE) to enhance the activity of human‐derived immunotoxins. We are developing a recombinant immunotoxin based on human DNase I, using a furin‐cleavable linker and KDEL ER retention sequence adapted from the PE intoxication strategy. We expect this construct to have low immunogenicity, high specificity for dividing cells, and enhanced toxicity relative to the unmodified DNase I construct.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of aromatic residue–controlled protein retention in the endoplasmic reticulum of Saccharomyces cerevisiae

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within Saccharomyces cerevisiae cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.

Novel Local RYR1 Junctional Calcium Responses and Dynamics by Improved Calcium Sensor Catcher

The precise spatio-temporal characteristics of Ca2+ transient are critical for the physiological regulation of Ca2+-dependent signaling processes. Because determining Ca2+ dynamics occurring in the myoplasm and sarcoplasmic reticulum (SR) under normal conditions is essential in order to fully understand changes that may occur under stressful or pathological conditions, we designed a new generation of Ca2+ sensors. Here, we report the optimization of a genetically-encoded Ca2+ sensor called “CatchER” which exhibits rapid kinetics and specific targeting to the endoplasmic reticulum (ER). We also describe the characteristics of CatchER-T’, a sensor variant with significantly improved specificities, including enhanced fluorescence at 37°C and a greater calcium dependent dynamic range. CatchER-T’ is targeted to the ER via the signal peptide of calreticulin and the ER retention sequence KDEL. Differential drug induced Ca2+ releases and ER refilling are observed for various cell types. Furthermore in electroporated mouse flexor digitorum brevis (FDB) muscle fibers, targeting CatchER-T’ close to the RyR1 channel via a sequence obtained from the junctional SR protein JP-45, revealed that local Ca2+ release via electrical stimulation is significantly greater and faster than that occurring in the SR lumen as assessed using CatchER-T’, i.e. the probe targeted to the lumen of the longitudinal SR. The impact of local ER/SR calcium dynamics in controlling cellular signaling and excitation contraction coupling will be presented.

Accumulation of foreign polypeptides to rice seed protein body type I using prolamin portion sequences

Key messageRice prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of prolamins that must accumulate in PB-Is.Rice seed prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice prolamins, we compared 13a and 13b prolamins because the amino acid sequences of these prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.