Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus.

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

A vital sugar code for ricin toxicity.

Ricin is one of the most feared bioweapons in the world due to its extreme toxicity and easy access. Since no antidote exists, it is of paramount importance to identify the pathways underlying ricin toxicity. Here, we demonstrate that the Golgi GDP-fucose transporter Slc35c1 and fucosyltransferase Fut9 are key regulators of ricin toxicity. Genetic and pharmacological inhibition of fucosylation renders diverse cell types resistant to ricin via deregulated intracellular trafficking. Importantly, cells from a patient with SLC35C1 deficiency are also resistant to ricin. Mechanistically, we confirm that reduced fucosylation leads to increased sialylation of Lewis X structures and thus masking of ricin-binding sites. Inactivation of the sialyltransferase responsible for modifications of Lewis X (St3Gal4) increases the sensitivity of cells to ricin, whereas its overexpression renders cells more resistant to the toxin. Thus, we have provided unprecedented insights into an evolutionary conserved modular sugar code that can be manipulated to control ricin toxicity.

Elongated and Shortened Peptidomimetic Inhibitors of the Proprotein Convertase Furin.

Novel elongated and shortened derivatives of the peptidomimetic furin inhibitor phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide were synthesized. The most potent compounds, such as Nα (carbamidoyl)Arg-Arg-Val-Arg-4-amidinobenzylamide (Ki =6.2 pm), contain additional basic residues at the N terminus and inhibit furin in the low-picomolar range. Furthermore, to decrease the molecular weight of this inhibitor type, compounds that lack the P5 moiety were prepared. The best inhibitors of this series, 5-(guanidino)valeroyl-Val-Arg-4-amidinobenzylamide and its P3 tert-leucine analogue displayed Ki values of 2.50 and 1.26 nm, respectively. Selected inhibitors, together with our previously described 4-amidinobenzylamide derivatives as references, were tested in cell culture for their activity against furin-dependent infectious pathogens. The propagation of the alphaviruses Semliki Forest virus and chikungunya virus was strongly inhibited in the presence of selected derivatives. Moreover, a significant protective effect of the inhibitors against diphtheria toxin was observed. These results confirm that the inhibition of furin should be a promising approach for the short-term treatment of acute infectious diseases.

The anti-tumor drug 2-hydroxyoleic acid (Minerval) stimulates signaling and retrograde transport.

2-hydroxyoleic acid (OHOA, Minerval®) is an example of a substance used for membrane lipid therapy, where the cellular membranes rather than specific proteins constitute the therapeutical target. OHOA is thought to mediate its anti-tumor effect by affecting the biophysical properties of membranes, which leads to altered recruitment and activation of amphitropic proteins, altered cellular signaling, and eventual cell death. Little is known about the initial signaling events upon treatment with OHOA, and whether the altered membrane properties would have any impact on the dynamic intracellular transport system. In the present study we demonstrate that treatment with OHOA led to a rapid release of intracellular calcium and activation of multiple signaling pathways in HeLa cells, including the PI3K-AKT1-MTOR pathway and several MAP kinases, in a process independent of the EGFR. By lipidomics we confirmed that OHOA was incorporated into several lipid classes. Concomitantly, OHOA potently increased retrograde transport of the plant toxin ricin from endosomes to the Golgi and further to the endoplasmic reticulum. The OHOA-stimulated ricin transport seemed to require several amphitropic proteins, including Src, phospholipase C, protein kinase C, and also Ca2+/calmodulin. Interestingly, OHOA induced a slight increase in endosomal localization of the retromer component VPS35. Thus, our data show that addition of a lipid known to alter membrane properties not only affects signaling, but also intracellular transport.

Cellular effects of fluorodeoxyglucose: Global changes in the lipidome and alteration in intracellular transport.

2-fluoro-2-deoxy-D-glucose (FDG), labeled with 18F radioisotope, is the most common imaging agent used for positron emission tomography (PET) in oncology. However, little is known about the cellular effects of FDG. Another glucose analogue, 2-deoxy-D-glucose (2DG), has been shown to affect many cellular functions, including intracellular transport and lipid metabolism, and has been found to improve the efficacy of cancer chemotherapeutic agents in vivo. Thus, in the present study, we have investigated cellular effects of FDG with the focus on changes in cellular lipids and intracellular transport. By quantifying more than 200 lipids from 17 different lipid classes in HEp-2 cells and by analyzing glycosphingolipids from MCF-7, HT-29 and HBMEC cells, we have discovered that FDG treatment inhibits glucosylceramide synthesis and thus reduces cellular levels of glycosphingolipids. In addition, in HEp-2 cells the levels and/or species composition of other lipid classes, namely diacylglycerols, phosphatidic acids and phosphatidylinositols, were found to change upon treatment with FDG. Furthermore, we show here that FDG inhibits retrograde Shiga toxin transport and is much more efficient in protecting cells against the toxin than 2DG. In summary, our data reveal novel effects of FDG on cellular transport and glycosphingolipid metabolism, which suggest a potential clinical application of FDG as an adjuvant for cancer chemotherapy.

A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.

JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin's enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin's bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells.

Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin's Enzymatic Subunit Interfere with Intracellular Toxin Transport.

Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin's enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin's egress from EEA-1(+) and Rab7(+) vesicles and enhanced toxin accumulation in LAMP-1(+) vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA.

Cross-linking of glycosphingolipids at the plasma membrane: consequences for intracellular signaling and traffic.

Glycosphingolipids (GSLs) are predominantly found in the outer leaflet of the plasma membrane, where they play a role in important processes such as cell adhesion, migration and signaling. However, by which mechanisms GSLs regulate these processes remains elusive. In this study, we therefore took advantage of the fact that some GSLs also serve as receptors for certain protein toxins, which rely on receptor binding for internalization and intoxication. Here, we demonstrate that Shiga and cholera toxins, which both possess multivalent GSL-binding capacity, induce dissociation of the cytosolic cPLA2α-AnxA1 complex in HeLa and HMEC-1 cells. The dissociation is mediated through an increase in cytosolic calcium levels and activation of the tyrosine kinase Syk. Ricin, a protein toxin that does not cross-link surface molecules, has no effect on the same complex. Importantly, we find that antibody-mediated cross-linking of Gb3 and GM1, the GSL receptors for Shiga and cholera toxin, respectively, also induces dissociation. These data demonstrate that cross-linking of GSLs at the plasma membrane mediates the intracellular signaling events resulting in dissociation of the complex. After dissociation, cPLA2α and AnxA1 are translocated to intracellular membranes where they are known to function in regulating membrane transport processes. In conclusion, we have characterized a novel mechanism for cell surface-induced initiation of intracellular signaling and transport events.

Novel actions of 2-deoxy-D-glucose: protection against Shiga toxins and changes in cellular lipids.

2-Deoxy-D-glucose (2DG) is a structural analogue of glucose with well-established applications as an inhibitor of glycolysis and N-glycosylation. Importantly, 2DG has been shown to improve the efficacy of several cancer chemotherapeutic agents in vivo and thus it is in clinical studies in combination with chemotherapy and radiotherapy. However, although 2DG has been demonstrated to modulate many cellular functions, including autophagy, apoptosis and cell cycle control, little is known about the effects of 2DG on intracellular transport, which is of great importance when predicting the effects of 2DG on therapeutic agents. In addition to proteins, lipids play important roles in cellular signalling and in controlling cellular trafficking. We have, in the present study, investigated the effects of 2DG on cellular lipid composition and by use of protein toxins we have studied 2DG-mediated changes in intracellular trafficking. By quantifying more than 200 individual lipid species from 17 different lipid classes, we have found that 2DG treatment changes the levels and/or species composition of several lipids, such as phosphatidylinositol (PI), diacylglycerol (DAG), cholesteryl ester (CE), ceramide (Cer) and lysophospho-lipids. Moreover, 2DG becomes incorporated into the carbohydrate moiety of glycosphingolipids (GSLs). In addition, we have discovered that 2DG protects cells against Shiga toxins (Stxs) and inhibits release of the cytotoxic StxA1 moiety in the endoplasmic reticulum (ER). The data indicate that the 2DG-induced protection against Stx is independent of inhibition of glycolysis or N-glycosylation, but rather mediated via the depletion of Ca(2+) from cellular reservoirs by 2DG. In conclusion, our results reveal novel actions of 2DG on cellular lipids and Stx toxicity.

Antibody-mediated inhibition of ricin toxin retrograde transport.

Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin's binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. IMPORTANCE Ricin is the prototypic member of the AB family of medically important plant and bacterial toxins that includes cholera and Shiga toxins. Ricin is also a category B biothreat agent. Despite ongoing efforts to develop vaccines and antibody-based therapeutics against ricin, very little is known about the mechanisms by which antibodies neutralize this toxin. In general, it is thought that antibodies simply prevent toxins from attaching to cell surface receptors or promote their clearance through Fc receptor (FcR)-mediated uptake. In this report, however, we describe a neutralizing monoclonal antibody (MAb) against ricin's binding subunit (RTB) that not only associates with ricin after the toxin has bound to the cell's surface but actually enhances toxin uptake into host cells. Following endocytosis, the antibody-toxin complexes are then routed for degradation. The results of this study are important because they reveal a previously unappreciated role for B-subunit-specific antibodies in intracellular neutralization of ricin toxin.

Retrograde transport of protein toxins through the Golgi apparatus.

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.

Annexin A1 and A2: roles in retrograde trafficking of Shiga toxin.

Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA(2)), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA(2) activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA(2).

Role of phospholipase A(2) in retrograde transport of ricin.

Ricin is a protein toxin classified as a bioterror agent, for which there are no known treatment options available after intoxication. It is composed of an enzymatically active A-chain connected by a disulfide bond to a cell binding B-chain. After internalization by endocytosis, ricin is transported retrogradely to the Golgi and ER, from where the ricin A-chain is translocated to the cytosol where it inhibits protein synthesis and thus induces cell death. We have identified cytoplasmic phospholipase A(2) (PLA(2)) as an important factor in ricin retrograde transport. Inhibition of PLA(2) protects against ricin challenge, however the toxin can still be endocytosed and transported to the Golgi. Interestingly, ricin transport from the Golgi to the ER is strongly impaired in response to PLA(2) inhibition. Confocal microscopy analysis shows that ricin is still colocalized with the trans-Golgi marker TGN46 in the presence of PLA(2) inhibitor, but less is colocalized with the cis-Golgi marker GM130. We propose that PLA(2) inhibition results in impaired ricin transport through the Golgi stack, thus preventing it from reaching the ER. Consequently, ricin cannot be translocated to the cytosol to exert its toxic action.

Kallikrein 4 is a proliferative factor that is overexpressed in prostate cancer.

Kallikrein 4 (KLK4) is a member of the human tissue KLK family. Whereas all other KLKs are secreted proteins with extracellular functions, KLK4 is primarily localized to the nucleus, indicating that it has a different function compared with other members of the KLK family. In addition, KLK4 expression is highly enriched in the prostate and is regulated by androgens. Here, we studied the possible functional role of KLK4 in prostate cancer cells and examined its expression at the protein level in prostate cancer specimens. Consistent with its mRNA expression, KLK4 protein is significantly overexpressed in malignant prostate compared with normal prostate. KLK4 expression is predominantly in the nucleus of basal cells in the prostate epithelium in keeping with its distribution in prostate cancer cells in vitro. Furthermore, adenovirus-mediated expression of KLK4 dramatically induces proliferation of prostate cancer cells, at least in part through significant alterations in cell cycle regulatory gene expression. Consistent with these data, small interfering RNA-mediated knockdown of endogenous KLK4 in LNCaP prostate cancer cells inhibits cell growth. These data identify KLK4 as the first member of the KLK family that is a proliferative factor with effects on gene expression and indicate that it may have an important role in prostate cancer development and progression.

Kallikrein 4 expression is up-regulated in epithelial ovarian carcinoma cells in effusions.

We immunohistochemically analyzed kallikrein 4 protein (hK4) expression in patients with epithelial ovarian carcinoma (181 malignant effusions and 103 solid carcinoma lesions). Expression of hK4 was also studied in 32 effusions using immunoblotting. Carcinoma cells expressed hK4 in 144 (79.6%) of 181 effusions and 85 (82.5%) of 103 solid tumors. Expression was seen in 51% or more of tumor cells in 70 effusions but often was limited to 5% or fewer cells in solid tumors (P = .009, primary tumors vs effusions; P = .002, metastases vs effusions). Immunoblotting showed hK4 expression in 31 of 32 specimens. Stromal cell hK4 expression, seen in 48 (46.6%) of 103 lesions, was significantly higher in primary tumors than metastases (26/43 vs 22/60, P = .019). hK4 expression in tumor cells was significantly lower in International Federation of Gynecology and Obstetrics stage IV than stage III tumors (P = .004, all lesions; P = .012, primary tumors). hK4 expression in carcinoma cells was associated with longer overall survival (not significant; P = .14, peritoneal effusions). hK4 is expressed widely in ovarian carcinoma; levels in carcinoma cells are highest in effusions, which might be related to loss of stromal contribution and/or altered microenvironment. hK4 expression in carcinoma cells of effusions or solid tumors does not predict survival.

Kallikrein 4 is associated with paclitaxel resistance in ovarian cancer.

OBJECTIVE: Carcinoma of the ovary is the most fatal malignancy of the female genital tract in western countries. The effectiveness of chemotherapy is limited by the acquisition of drug resistance. Several members of the kallikrein (KLK) family were recently shown to be expressed in ovarian cancer and implicated in disease prognosis. One of these is KLK4, whose increased mRNA expression was identified as a poor prognostic marker in ovarian cancer. The goal of this study was to investigate if KLK4 protein (hK4) is expressed in ovarian carcinoma lesions and if it is associated with paclitaxel resistance. METHODS: Immunohistochemistry was used to assess hK4 expression. The 126 lesions were from a cohort of 46 patients with platinum-resistant tumors, which were treated with weekly paclitaxel for recurrent disease. The overall response rate to weekly paclitaxel was 52% (24 of 46) and for patients with tumors resistant to standard three weekly paclitaxel treatment 48% (16 of 33). RESULTS: Immunohistochemistry indicated that 85% (79 of 93) of lesions from paclitaxel-resistant patients expressed hK4, while only 61% (20 of 33) of lesions were positive for hK4 from paclitaxel sensitive patients. Statistical analysis showed that the intensity of hK4 staining was significantly associated with paclitaxel resistance (P = 0.005), whereas hK4 staining extent showed marginal significance (P = 0.05), but was significantly correlated with higher histological grade (P < 0.001). CONCLUSION: These data show that hK4 is expressed in ovarian cancer and suggest that hK4 expression might be a predictive marker for paclitaxel resistance.

Kallikrein 4 is a predominantly nuclear protein and is overexpressed in prostate cancer.

Kallikreins (KLKs) are highly conserved serine proteases that play key roles in a variety of physiological and pathological processes. KLKs are secreted proteins that have extracellular substrates and function. For example, prostate-specific antigen (or KLK3) is a secreted protein that is widely used as a diagnostic marker for prostate cancer. KLK4 is a recently identified member of the kallikrein family that is regulated by androgens and is highly specific to prostate for expression. Here, we show that the gene product of KLK4, hK4, is the first member of the KLK family that is intracellularly localized. We provide strong evidence that the previously assigned first exon that was predicted to code for a signal peptide that would target hK4 for secretion is not part of the physiologically relevant form of KLK4 mRNA. In addition to detailed mapping of the KLK4 mRNA 5' end by RT-PCR, this conclusion is supported by predominantly nuclear localization of the hK4 protein in the cell, documented by both immunofluorescence and cell fractionation experiments. Furthermore, in addition to androgens, hK4 expression is regulated by estrogen and progesterone in prostate cancer cells. Finally, in situ hybridization on normal and hyperplastic prostate samples in tissue microarrays indicate that KLK4 is predominantly expressed in the basal cells of the normal prostate gland and overexpressed in prostate cancer. These data suggest that KLK4 has a unique structure and function compared with other members of the KLK family and may have a role in the biology and characterization of prostate cancer.