Christoph Arenz

Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology.

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism. In acidic environments Hsp70 binds with high affinity and specificity to BMP, thereby facilitating the BMP binding and activity of acid sphingomyelinase (ASM). The inhibition of the Hsp70–BMP interaction by BMP antibodies or a point mutation in Hsp70 (Trp90Phe), as well as the pharmacological and genetic inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann–Pick disease (NPD) A and B—severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM—is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds that enter the lysosomal lumen by the endocytic delivery pathway.

Transformation-Associated Changes in Sphingolipid Metabolism Sensitize Cells to Lysosomal Cell Death Induced by Inhibitors of Acid Sphingomyelinase

Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert multidrug resistance. Their cancer selectivity is associated with transformation-associated reduction in ASM expression and subsequent failure to maintain sphingomyelin hydrolysis during drug exposure. Taken together, these data identify ASM as an attractive target for cancer therapy.

Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer.

The microprocessor complex mediates intranuclear biogenesis of precursor microRNAs from the primary microRNA transcript. Extranuclear, mature microRNAs are incorporated into the RNA‐induced silencing complex (RISC) before interaction with complementary target mRNA leads to transcriptional repression or cleavage. In this study, we investigated the expression profiles of the microprocessor complex subunit DiGeorge syndrome critical region gene 8 (DGCR8) and the RISC components argonaute‐1 (AGO1), argonaute‐2 (AGO2), as well as double‐stranded RNA‐binding proteins PACT, TARBP1, and TARBP2 in epithelial skin cancer and its premalignant stage. Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional), from healthy skin sites (intraindividual controls), and from healthy skin sites in a healthy control group (n = 16; interindividual control). The DGCR8, AGO1, AGO2, PACT, TARBP1, and TARBP2 mRNA expression levels were detected by quantitative real‐time reverse transcriptase polymerase chain reaction. The DGCR8, AGO1, AGO2, PACT, and TARBP1 expression levels were significantly higher in the AK, BCC, and SCC groups than the healthy controls (P < 0.05). There was no significant difference in the TARBP2 expression levels between groups (P > 0.05). This study indicates that major components of the miRNA pathway, such as the microprocessor complex and RISC, are dysregulated in epithelial skin cancer.

Potent and Selective Inhibition of Acid Sphingomyelinase by Bisphosphonates

The acid sphingomyelinase (aSMase) is emerging as an important drug target for a variety of diseases. Inhibition of aSMase prevents bacterial infections in a rat model of cystic fibrosis and formation of acute lung injury (ALI) elicited by endotoxin, acid instillation, or platelet-activating factor (PAF). Moreover, aSMase is essential for infection of non-phagocytotic cells with Neisseria gonorrhoeae and formation of pulmonary emphysema. Pharmacological or genetic inhibition of aSMase prevents apoptosis and degeneration of liver cells in a mouse model for Wilson s disease. In addition, there are several reports that aSMase significantly contributes to the formation of atherosclerotic plugs. This promising progress in aSMase research, based on sophisticated animal models and cultured cells from patients, is thwarted, however, by the lack of potent and selective inhibitors of this enzyme. Phosphatidylinositol-3,5-bisphosphate (PtdIns3,5P2), to date the most potent inhibitor (KM = 0.53 mm), is not suitable for cell culture studies, because of its fivefold negative charge and its two long fatty acid chains which cause it to stack in cellular membranes. Last but not least, this inhibitor is labile towards phospholipases A1, A2, C, and D and phosphoinositide phosphatases. The aSMase is a soluble lysosomal sphingolipid hydrolase, which constitutively degrades sphingomyelin from internalized membrane fragments. Upon stimulation, however, a portion of this enzyme can be found on the outer side of the plasma membrane. This membrane-associated enzyme shows biochemical activity in serum and urine and has been termed secretory sphingomyelinase (sSMase), although it is virtually identical to the lysosomal variant. Its activity is elevated in several diseases. The secretory form of aSMase is believed to play an important role in signal transduction, since it alters the composition of the plasma membrane within putative sphingolipidand cholesterol-rich membrane microdomains. These so-called “lipid rafts” have been suggested to act as “signaling platforms”, and there is significant evidence that the cleavage of sphingomyelin to ceramide can dramatically alter the biophysical properties of the putative rafts. In addition, it is well established that ceramide is a potent inductor of apoptosis, which is the main reason for cell degeneration in many of the diseases mentioned above. However, it is unknown whether ceramide acts by remodeling the plasma membrane or by interacting with proteins like cathepsin B, which is involved in cellular signaling. Beside aSMase, two cytosolic, magnesium-dependent and membrane-bound neutral sphingomyelinases (nSMase1 and nSMase2) and an alkaline sphingomyelinase are known, whose cellular function is rather unclear. Recently nSMase has been shown to be essential for the formation of exosomes, lipid vesicles that play a key role in the infection by retroviruses. In contrast to aSMase, there are some potent small-molecule inhibitors for nSMase. Our attempts at synthesizing phosphonate analogues of PtdIns3,5P2 as potential inhibitors of aSMase yielded only moderately active substances. However, we also gained access to a collection of (bis)phosphonates that had been synthesized in the GDR Academy of Sciences and that contained some compounds that are structurally related to our phosphoinositide analogues. When we initially tested these substances at a concentration of 20 mm, we were surprised that some of them were potent inhibitors of aSMase (Tables 1 and 2). Among these substances, a-amino-

Development of an assay for the intermembrane transfer of cholesterol by Niemann-Pick C2 protein.

Abstract Niemann-Pick type C disease is an inherited fatal disorder characterized by the accumulation of unesterified cholesterol and other lipids in the endosomal/lysosomal compartment. Two independent genes responsible for this neurodegenerative disorder have been identified, but the precise functions of the encoded Niemann-Pick C1 (NPC1) and C2 (NPC2) proteins are not yet known. We developed a cell-free assay for measuring intermembrane lipid transport and examined the ability of bovine NPC2 (bNPC2) for intermembrane cholesterol transfer. NPC2 specifically extracts cholesterol from phospholipid bilayers and catalyzes intermembrane transfer to acceptor vesicles in a dose- and time-dependent manner. This transfer activity is dependent on temperature, pH, ionic strength, lipid composition of the model membranes, and the ratio of donor to acceptor vesicles. In model membranes, the presence of the lysosomal anionic phospholipids bis(monooleoylglycero)phosphate and phosphatidyl inositol significantly stimulated cholesterol transfer by NPC2, whereas bis(monomyristoylglycero)phosphate, phosphatidyl serine, and phosphatidic acid had no effect. Moreover, ceramide stimulated cholesterol transfer slightly, whereas sphingomyelin reduced cholesterol transfer rates. With our assay system we identified for the first time the ability of other lysosomal proteins, most notably the GM2-activator protein, to mediate intermembrane cholesterol transfer. This assay system promises to be a valuable tool for further quantitative and mechanistic studies of protein-mediated lipid transfer.

Phosphatidylinositol-3,5-bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Abstract Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-?-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P[2]), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P[2] is 0.53 M as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 uM, PtdIns3,5P[2] neither decreased plasma membrane associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases ?-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.

Synthesis and biochemical investigation of scyphostatin analogues as inhibitors of neutral sphingomyelinase.

The sphingolipid ceramide is considered to be an important intracellular mediator. However, many aspects of its action and the role of several different ceramide generating sphingomyelinases are still unclear. Recently, we reported on the synthesis of the first selective irreversible inhibitor of the neutral sphingomyelinase (N-SMase), as well as the identification of Manumycin A and some of its analogues as irreversible inhibitors of N-SMase. For the development of pharmacologically interesting competitive inhibitors of N-SMase, structure-activity studies are essential. Herein we show the synthesis and enzymatic investigation of two scyphostatin analogues 3a and 3b, revealing the importance of the primary hydroxy group in compound 2 for N-SMase inhibition.

Long Endogenous dsRNAs Can Induce Complete Gene Silencing in Mammalian Cells and Primary Cultures

Recently, double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) has rapidly developed to a powerful instrument for specific silencing of gene expression in several organisms, including Caenorhabditis elegans, Drosophila melanogaster, and plants. The finding that synthetic small interfering RNAs (siRNAs) of 21 nt as well as stable, endogenously expressed, large dsRNA are suited to specifically induce gene silencing in mammalian cells offered the possibility of expanding this technique to mammalian systems. In this work, we engineered stably transfected human cells that express large dsRNAs mediating specific posttranscriptional silencing of genes. We used this technique to specifically silence genes coding for glucosylceramide synthase (GCS), the sphingolipid activator protein precursor (SAP), and glucocerebrosidase (GBA), all implicated in glycosphingolipid metabolism. From a 1600-bp inverted repeat DNA template, a dsRNA of 800 bp is expressed and predicted to mediate the specific suppression of the corresponding gene by RNAi. Remarkably, we were able to use this method to achieve complete inhibition of those genes we targeted in different cultured human cell lists. These findings testify to the generality of RNAi application in suppressing gene expression in mammalian cells.

Small Molecule Inhibitors of Acid Sphingomyelinase

Despite of the importance of the acid sphingomyelinase for sphingomyelin homeostasis and sphingolipid signalling, potent and selective inhibitors for this enzyme are rare. An increasing set of data on the inhibition of acid sphingomyelinase in different disease models using indirect inhibitors has been generated and strongly implies acid sphingomyelinase as an emerging drug target. Very recently, some new and promising inhibitors from different substance classes have been developed. In this review, previous and current developments in the field are summarized.

Heat shock protein–based therapy as a potential candidate for treating the sphingolipidoses

Increasing Hsp70 expression in lysosomes using the small-molecule arimoclomol ameliorates pathology in several animal models of sphingolipidoses. Heat shock protein to the rescue The sphingolipidoses constitute a major subgroup of lysosomal storage diseases, a class of inherited metabolic disorders characterized by severe systemic and neurological problems. Few therapeutic options exist for treating these disorders. Kirkegaard et al. now demonstrate that increasing the expression of the molecular chaperone HSP70 through administration of either recombinant human HSP70 or the clinically tested, orally available small-molecule arimoclomol ameliorated disease manifestations, including brain pathology, in several different animal models of sphingolipidoses. Lysosomal storage diseases (LSDs) often manifest with severe systemic and central nervous system (CNS) symptoms. The existing treatment options are limited and have no or only modest efficacy against neurological manifestations of disease. We demonstrate that recombinant human heat shock protein 70 (HSP70) improves the binding of several sphingolipid-degrading enzymes to their essential cofactor bis(monoacyl)glycerophosphate in vitro. HSP70 treatment reversed lysosomal pathology in primary fibroblasts from 14 patients with eight different LSDs. HSP70 penetrated effectively into murine tissues including the CNS and inhibited glycosphingolipid accumulation in murine models of Fabry disease (Gla−/−), Sandhoff disease (Hexb−/−), and Niemann-Pick disease type C (Npc1−/−) and attenuated a wide spectrum of disease-associated neurological symptoms in Hexb−/− and Npc1−/− mice. Oral administration of arimoclomol, a small-molecule coinducer of HSPs that is currently in clinical trials for Niemann-Pick disease type C (NPC), recapitulated the effects of recombinant human HSP70, suggesting that heat shock protein–based therapies merit clinical evaluation for treating LSDs.