Carboxy-terminal KDEL Research Articles

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants.

Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor—staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the β-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the β-phaseolin promoter.

Metformin Suppresses the GRP78-Dependent Delivery of Aggresomes to Autophagosomes to Enhance the Anti-Myeloma Benefit of Proteasome Inhibition

Background:Functional blockade of the ubiquitin (Ub)+proteasome system (UPS) using the small molecule bortezomib represents a remarkable bench-to-bedside success that has improved the outcome of patients diagnosed with multiple myeloma (MM). However, certain patients do not respond to bortezomib and those that do inevitably develop resistance through mechanisms that remain elusive and despite all currently available therapies, MM remains invariably fatal. While bortezomib disrupts protein homeostasis leading to tumor cell death but also activates pro-survival aggresome and autophagosome formation as compensatory protein clearance mechanisms to promote the emergence of drug resistance, tumor survival and disease relapse. While the therapeutic benefit of targeting the proteasome is unchallenged, more precise modalities that do not induce drug resistance are urgently needed. These studies were performed to identify pharmacologics that inhibited the autophagy pathway and to uncover their mechanisms of action as a means to synergistically enhance the cytotoxic effect of bortezomib for myeloma treatment.Methods:High-throughput screening (HTS) was performed to identify FDA-approved pharmacologic agents and bioactive molecules that prevented bortezomib-mediated induction of autophagosomes. Confocal microscopy combined with novel, dye-based fluorescent methods were used to visualize and quantitate aggresomes and autophagosomes. The effect of pharmacologics on aggresome and autophagosome formation was then determined. The effects of metformin, alone or combined with bortezomib were evaluated using MM cell lines, myeloma patient bone marrow-derived tumor cells and myeloma cells xenotransplanted into mouse models.Results:Bortezomib inhibits the UPS to promote the unwanted intracellular accumulation of ubiquitinated proteins that form aggregates dispersed throughout the cytoplasm. These aggregates are then coalesced into perinuclear aggresomes and subsequently disposed through autophagosome formation and the lysosomal pathway. Bortezomib treatment of myeloma cells increased levels of the endoplasmic reticulum (ER) molecular chaperone Glucose-Regulated Protein 78 (GRP78), a critical regulator of the unfolded protein response (UPR) and autophagosome formation. HTS revealed that the anti-diabetic biguanide metformin inhibited bortezomib-mediated autophagosome formation. Co-treatment with metformin and bortezomib increased the level of proteotoxic aggresomes but suppressed GRP78 and autophagosome formation. Metformin synergistically enhanced the anti-proliferative effect of bortezomib in myeloma cells and bone marrow-derived patient PCs. Co-treatment with metformin and proteasome inhibitors also overcame drug-resistance. shRNA-mediated knockdown of HSPA5, which encodes GRP78, similarly enhanced bortezomib-induced aggresome formation and blocked autophagosome formation. A GRP78 mutant that lacked the carboxy-terminal KDEL ER-retention motif also led to bortezomib-induced aggresomes without autophagosomes. Metformin and bortezomib together reduced myeloma xenotransplant growth in mice more effectively than either agent administered as single-agent.Conclusions:The ER chaperone GRP78 facilitates aggresome clearance by promoting autophagosome formation. Although bortezomib alone promotes GRP78 induction and aggresome formation, metformin suppresses GRP78 to uncouple the cytotoxic aggresomes from the autophagy pathway and to enhance the anti-myeloma effect of bortezomib. The results support the pharmacologic repositioning of metformin in combination with bortezomib as an effective anti-myeloma strategy. The molecular chaperone GRP78 holds promise as a target for cancer treatment as well as for neurodegenerative diseases in which intracellular protein aggregates are central to disease pathogenesis DisclosuresNo relevant conflicts of interest to declare.

Partial redistribution of the Autographa californica nucleopolyhedrovirus chitinase in virus-infected cells accompanies mutation of the carboxy-terminal KDEL ER-retention motif.

During virus infection of insect cells, the Autographa californica nucleopolyhedrovirus chitinase is localized primarily within the endoplasmic reticulum (ER), which is consistent with the presence of a carboxy-terminal ER retention motif (KDEL). Release of chitinase into the extracellular medium appears to be concomitant with terminal cell lysis, rather than by active secretion. In this study, we have shown that mutation of the KDEL motif induces a partial redistribution of the chitinase at both early and late times post-infection. Deletion of the KDEL motif or substitution with glycine residues allowed chitinase to move through the secretory pathway, accumulating to detectable levels in the extracellular medium by 24 h post-infection; more than 48 h prior to cell lysis. Deletion of the KDEL motif did not compromise enzyme activity, with the modified enzyme exhibiting characteristic endo- and exo-chitinolytic activity. Trichoplusia ni larvae infected with the modified virus were found to liquefy approximately 24 h earlier than larvae infected with a control virus in which the chitinase KDEL motif had not been deleted.

PROTEIN RETENTION IN THE ENDOPLASMIC RETICULUM OF INSECT CELLS IS NOT COMPROMISED BY BACULOVIRUS INFECTION

High level expression of the major auxin-binding protein (ABP1) from maize ( Zea maysL.) has been used to demonstrate that the machinery for retaining proteins in the endoplasmic reticulum (ER) of insect cells functions efficiently throughout the baculovirus infection cycle. Immuno-localization showed wild-type ABP1 (ABP1-KDEL) to be targeted to the lumen of the ER, in accordance with its signal peptide and carboxyterminal KDEL ER-retention signal. The protein accumulated in dilations of the ER, and none was detected at the cell surface. Immunoblotting of concentrated culture medium confirmed that ABP1-KDEL was not secreted at a detectable level. In contrast, when the carboxyterminus was mutated to KEQL, secretion of the baculovirus-expressed protein was readily detected. Immunolocalization and immunoblotting demonstrated that a high proportion of the ABP1-KEQL protein was secreted at the cell surface and into the culture medium. The data demonstrate that the ER of insect cells has a great capacity to retain proteins and that this property is largely unaffected by the cellular disruption caused by baculovirus replication.

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants.

Gene constructs were designed to test the effect of the endoplasmic reticulum (ER)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35S promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDEL-coding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steady-state levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. Immunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin. It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

N-Ac-KDEL induces rapid alterations in endoplasmic reticulum structure

KDEL (i.e., Lysine-Aspartate-Glutamate-Leucine) is a tetrapeptide motif that serves as a retention sequence at the carboxy-terminus of proteins which reside within the ER lumen. A specific receptor located in or near the cis-Golgi recognizes proteins bearing carboxyterminal KDEL and mediates their return to the ER. We reasoned that it may be possible to saturate the KDEL receptor with a hapten, thereby competitively inhibiting its function in living cells. We hypothesized that if we blinded the receptor with an excess of hapten then the intermediate compartment should be unable to differentiate between polypeptides containing carboxyterminal KDEL from those that do not. If the receptor could be blinded, then KDEL-polypeptides such as BiP should be secreted.

Retention of phytohemagglutinin with carboxyterminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum

Soluble proteins that reside in the lumen of the endoplasmic reticulum are known to have at their carboxyterminus the tetrapeptides KDEL or HDEL. In yeast and mammalian cells, these tetrapeptides function as endoplasmic reticulum (ER)-retention signals. To determine the effect of an artificially-introduced KDEL sequence at the exact carboxyterminus of a plant secretory protein, we modified the gene of the vacuolar protein phytohemagglutinin-L (PHA) so that the amino-acid sequence would end in LNKDEL rather than LNKIL, and expressed the modified gene in transgenic tobacco with a seed-specific promoter. Analysis of the glycans of PHA showed that most of the control PHA had one endoglycosidase H-sensitive and one endoglycosidase H-resistant glycan, indicating that it had been processed in the Golgi complex. On the other hand, a substantial portion of the PHA-KDEL (about 75% at mid-maturation and 50% in mature seeds) had two endoglycosidase H-sensitive glycans. Phytohemagglutinin with two endoglycosidase H-sensitive glycans is normally found in the ER. Using immunocytochemistry we found that a substantial portion of the PHA-KDEL was present in the ER or accumulated in the nuclear envelope while the remainder was found in the protein storage vacuoles (protein bodies). We interpret these data to indicate that carboxyterminal KDEL functions as an ER retention-retardation signal and causes protein to accumulate in the nuclear envelope as well as in the ER. The incomplete ER retention of this protein which is modified at the exact carboxyterminus may indicate that structural features other than carboxyterminal KDEL are important if complete ER retention is to be achieved.Mention of trademark, proprietary product, or vendor, does not constitute a guarantee or warrenty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal

Monoclonal antibodies were raised against antibodies to distinct carboxy-terminal KDEL sequences of two soluble, resident endoplasmic reticulum proteins. These anti-idiotype reagents recognize an intrinsic membrane protein with characteristics expected of a receptor responsible for the recognition and return of resident proteins to the endoplasmic reticulum.