Retinol Esterification by DGAT1 Is Essential for Retinoid Homeostasis in Murine Skin

Retinoic acid (RA) is a potent signaling molecule that is essential for many biological processes, and its levels are tightly regulated by mechanisms that are only partially understood. The synthesis of RA from its precursor retinol (vitamin A) is an important regulatory mechanism. Therefore, the esterification of retinol with fatty acyl moieties to generate retinyl esters, the main storage form of retinol, may also regulate RA levels. Here we show that the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol acyltransferase (ARAT) in murine skin. When dietary retinol is abundant, DGAT1 deficiency results in elevated levels of RA in skin and cyclical hair loss; both are prevented by dietary retinol deprivation. Further, DGAT1-deficient skin exhibits enhanced sensitivity to topically administered retinol. Deletion of the enzyme specifically in the epidermis causes alopecia, indicating that the regulation of RA homeostasis by DGAT1 is autonomous in the epidermis. These findings show that DGAT1 functions as an ARAT in the skin, where it acts to maintain retinoid homeostasis and prevent retinoid toxicity. Our findings may have implications for human skin or hair disorders treated with agents that modulate RA signaling.Regulation of cellular proliferation and differentiation of epithelial tissues is crucial in embryonic development and in adult homeostasis. Retinoic acid (RA)² is a major regulator of these processes (1) through its ability to serve as a ligand for RA nuclear receptors (RARs) (2). Since RA is such a potent signaling molecule, its levels must be tightly controlled. Indeed, excess RA is highly teratogenic during embryonic development and may be toxic to adult tissues (3). Further, RA is used therapeutically for skin disorders, such as acne and psoriasis, and certain cancers (4), but its uses are often limited by local and systemic toxicity. Thus, understanding how RA levels are regulated has important biological and clinical relevance.The synthesis of RA from its precursor retinol, or vitamin A, is a major node in the regulation of RA levels (5). To generate RA, retinol is oxidized in two sequential reactions, catalyzed by retinol and retinal dehydrogenases (5), whose activities regulate RA homeostasis. We hypothesized that the availability of retinol for these reactions may also be regulated by the balance between retinol and retinyl esters. Indeed, the majority of retinol in the body is stored as retinyl esters, which are concentrated in cytosolic lipid droplets of cells and serve as a local source of retinol. Retinyl esters are also stored in major organs, such as liver and white adipose tissue (WAT), from which retinol can be mobilized to supply other tissues during increased demand. Thus, retinol esterification may participate in regulating the retinol pool available for RA synthesis.Retinol esterification is carried out by two distinct enzymatic activities. One is mediated by lecithin:retinol acyltransferase (LRAT), which catalyzes the covalent joining of a fatty acyl moiety from lecithin (phosphatidylcholine) to retinol that is bound to cellular retinol-binding protein (CRBP) (6, 7). LRAT activity is crucial for maintaining tissue retinol stores. LRAT-null (Lrat^-/-) mice have severe reductions in hepatic and lung retinyl ester levels (8–10), which are accompanied by testicular hypoplasia/atrophy (9) and blindness (8). Retinyl ester levels are normal in WAT and several other tissues, indicating alternative mechanisms for retinol esterification (9, 10). This esterification is probably mediated in part by acyl CoA:retinol acyltransferase (ARAT) enzymes, which use fatty acyl-CoA and unbound retinol as substrates (11). Although many tissues exhibit ARAT activity (12), attempts to purify and clone an ARAT gene were unsuccessful, and thus molecular tools to study ARAT activity have been lacking. However, the enzyme encoded by Dgat1, an acyl CoA:diacylglycerol acyltransferase (DGAT), was recently reported to catalyze the ARAT reaction in vitro (13, 14). Moreover, several tissues of Dgat1^-/- mice had reduced ARAT activity, and retinol esterification was reduced in cultured murine embryonic fibroblasts lacking DGAT1 (14). Most recently, a study of Dgat1^-/- mice demonstrated a role for the enzyme in retinol absorption in the small intestine (15). Thus, accumulating evidence indicates that the retinol esterification activity of DGAT1 is of biological, and possibly clinical, importance.In the current study, we investigated whether retinol esterification by DGAT1 is important in murine skin. Dgat1^-/- mice exhibit a pleiotropic phenotype, which includes resistance to diet-induced obesity and altered energy metabolism but also includes prominent phenotypic findings in the skin (16–19). Retinoids play key roles in skin and hair biology (20), and excess retinoids induce epidermal hyperplasia, inhibit sebocyte proliferation and differentiation, and alter hair growth (21). Notably, the skin manifestations of Dgat1^-/- mice, which include alopecia and sebaceous gland atrophy (18), resemble those of retinoid toxicity (22, 23). Thus, we hypothesized that DGAT1 functions as an ARAT in murine skin and that the absence of DGAT1 alters retinoid homeostasis. In this study, we tested this hypothesis by examining retinoid metabolism in the skin of DGAT1-deficient mice.     

Prdm6 Is Essential for Cardiovascular Development In Vivo

Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice. Complete deletion of Prdm6 results in embryonic lethality due to cardiovascular defects associated with aberrations in vascular patterning. However, smooth muscle cells could be regularly differentiated from Prdm6-deficient embryonic stem cells and vascular smooth muscle cells were present and proliferated normally in Prdm6-deficient embryos. Conditional deletion of Prdm6 in the smooth muscle cell lineage using a SM22-Cre driver line resulted in perinatal lethality due to hemorrhage in the lungs. We thus identified Prdm6 as a factor that is essential for the physiological control of cardiovascular development.     

tmRNA Is Essential in Shigella flexneri

Nonstop mRNAs pose a challenge for bacteria, because translation cannot terminate efficiently without a stop codon. The trans-translation pathway resolves nonstop translation complexes by removing the nonstop mRNA, the incomplete protein, and the stalled ribosome. P1 co-transduction experiments demonstrated that tmRNA, a key component of the trans-translation pathway, is essential for viability in Shigella flexneri. tmRNA was previously shown to be dispensable in the closely related species Escherichia coli, because E. coli contains a backup system for trans-translation mediated by the alternative release factor ArfA. Genome sequence analysis showed that S. flexneri does not have a gene encoding ArfA. E. coli ArfA could suppress the requirement for tmRNA in S. flexneri, indicating that tmRNA is essential in S. flexneri because there is no functional backup system. These data suggest that resolution of nonstop translation complexes is required for most bacteria.     

HtrA Is Essential for Efficient Secretion of Recombinant Proteins by Lactococcus lactis

HtrA is a unique protease on the extracellular surface of Lactococcus lactis. It is known to take part in the proteolysis of many secreted recombinant proteins, and the mutation of htrA can lead to the complete stabilization of recombinant proteins. In this work, we have shown that htrA mutation also leads to significant reduction of the efficiency of recombinant-protein secretion. We also show that the level of HtrA can be lowered by the suppression of the acid tolerance response (ATR) in L. lactis. Instead of using an L. lactis htrA mutant, the reduction of the HtrA level in wild-type recombinant cultures of L. lactis by ATR suppression may serve as a better strategy for the production of secreted recombinant proteins.     

Fatty Acid Oxidation Is Essential for Egg Production by the Parasitic Flatworm Schistosoma mansoni

Schistosomes, parasitic flatworms that cause the neglected tropical disease schistosomiasis, have been considered to have an entirely carbohydrate based metabolism, with glycolysis playing a dominant role in the adult parasites. However, we have discovered a close link between mitochondrial oxygen consumption by female schistosomes and their ability to produce eggs. We show that oxygen consumption rates (OCR) and egg production are significantly diminished by pharmacologic inhibition of carnitine palmitoyl transferase 1 (CPT1), which catalyzes a rate limiting step in fatty acid β-oxidation (FAO) and by genetic loss of function of acyl CoA synthetase, which complexes with CPT1 and activates long chain FA for use in FAO, and of acyl CoA dehydrogenase, which catalyzes the first step in FAO within mitochondria. Declines in OCR and egg production correlate with changes in a network of lipid droplets within cells in a specialized reproductive organ, the vitellarium. Our data point to the importance of regulated lipid stores and FAO for the compartmentalized process of egg production in schistosomes.     

dRecQ4 Is Required for DNA Synthesis and Essential for Cell Proliferation in Drosophila

Background

The family of RecQ DNA helicases plays an important role in the maintenance of genomic integrity. Mutations in three of the five known RecQ family members in humans, BLM, WRN and RecQ4, lead to disorders that are characterized by predisposition to cancer and premature aging.

Methodology/Principal Findings

To address the in vivo functions of Drosophila RecQ4 (dRecQ4), we generated mutant alleles of dRecQ4 using the targeted gene knock-out technique. Our data show that dRecQ4 mutants are homozygous lethal with defects in DNA replication, cell cycle progression and cell proliferation. Two sets of experiments suggest that dRecQ4 also plays a role in DNA double strand break repair. First, mutant animals exhibit sensitivity to gamma irradiation. Second, the efficiency of DsRed reconstitution via single strand annealing repair is significantly reduced in the dRecQ4 mutant animals. Rescue experiments further show that both the N-terminal domain and the helicase domain are essential to dRecQ4 function in vivo. The N-terminal domain is sufficient for the DNA repair function of dRecQ4.

Conclusions/Significance

Together, our results show that dRecQ4 is an essential gene that plays an important role in not only DNA replication but also DNA repair and cell cycle progression in vivo.     

Dendritic Cell Cross-Priming Is Essential for Immune Responses to Listeria monocytogenes

Cross-presentation is now recognized as a major mechanism for initiating CD8 T cell responses to virus and tumor antigens in vivo. It provides an elegant mechanism that allows relatively few Dendritic cells (DCs) to initiate primary immune responses while avoiding the consumptive nature of pathogenic infection. CD8 T cells play a major role in anti-bacterial immune responses; however, the contribution of cross-presentation for priming CD8 T cell responses to bacteria, in vivo, is not well established. Listeria monocytogenes (Listeria) is the causative agent of Listeriosis, an opportunistic food-borne bacterial infection that poses a significant public health risk. Here, we employ a transgenic mouse model in which cross-presentation is uniquely inactivated, to investigate cross-priming during primary Listeria infection. We show that cross-priming deficient mice are severely compromised in their ability to generate antigen-specific T cells to stimulate MHC I-restricted CTL responses following Listeria infection. The defect in generation of Listeria-elicited CD8 T cell responses is also apparent in vitro. However, in this setting, the endogenous route of processing Listeria-derived antigens is predominant. This reveals a new experimental dichotomy whereby functional sampling of Listeria-derived antigens in vivo but not in vitro is dependent on cross-presentation of exogenously derived antigen. Thus, under normal physiological circumstances, cross-presentation is demonstrated to play an essential role in priming CD8 T cell responses to bacteria.     

The Lon Protease Is Essential for Full Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.     

Arabidopsis thaliana Glyoxalase 2-1 Is Required during Abiotic Stress but Is Not Essential under Normal Plant Growth

The glyoxalase pathway, which consists of the two enzymes, GLYOXALASE 1 (GLX 1) (E.C.: 4.4.1.5) and 2 (E.C.3.1.2.6), has a vital role in chemical detoxification. In Arabidopsis thaliana there are at least four different isoforms of glyoxalase 2, two of which, GLX2-1 and GLX2-4 have not been characterized in detail. Here, the functional role of Arabidopsis thaliana GLX2-1 is investigated. Glx2-1 loss-of-function mutants and plants that constitutively over-express GLX2-1 resemble wild-type plants under normal growth conditions. Insilico analysis of publicly available microarray datasets with ATTEDII, Mapman and Genevestigator indicate potential role(s) in stress response and acclimation. Results presented here demonstrate that GLX2-1 gene expression is up-regulated in wild type Arabidopsis thaliana by salt and anoxia stress, and by excess L-Threonine. Additionally, a mutation in GLX2-1 inhibits growth and survival during abiotic stresses. Metabolic profiling studies show alterations in the levels of sugars and amino acids during threonine stress in the plants. Elevated levels of polyamines, which are known stress markers, are also observed. Overall our results suggest that Arabidopsis thaliana GLX2-1 is not essential during normal plant life, but is required during specific stress conditions.     

Subcellular Origin of Sphingosine 1-Phosphate Is Essential for Its Toxic Effect in Lyase-deficient Neurons

Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient mice were used to study the toxicity of this potent sphingolipid metabolite in terminally differentiated postmitotic neurons. Based on earlier findings with the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate, we hypothesized that accumulation of S1P above a certain threshold induces neuronal apoptosis. The present studies confirmed this conclusion and further revealed that for S1P to induce apoptosis in lyase-deficient neurons it must also be produced by sphingosine-kinase2 (SK2). These conclusions are based on the finding that incubation of lyase-deficient neurons with either sphingosine or S1P results in a similar elevation in cellular S1P; however, only S1P addition to the culture medium induces apoptosis. This was not due to S1P acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was phosphorylated by the cells, as described before for cis-4-methylsphingosine. Although the cells produced S1P from both exogenously added sphingosine as well as sphingosine derived from exogenous S1P, the S1P from these two sources were not equivalent, because the former was primarily produced by SK1, whereas the latter was mainly formed by SK2 (as also was cis-4-methylsphingosine phosphate), based on studies in neurons lacking SK1 or SK2 activity. Thus, these investigations show that, due to the existence of at least two functionally distinct intracellular origins for S1P, exogenous S1P can be neurotoxic. In this model, S1P accumulated due to a defective lyase, however, this cause of toxicity might also be important in other cases, as illustrated by the neurotoxicity of cis-4-methylsphingosine phosphate.Sphingosine 1-phosphate (S1P)² is a potent lipid mediator that has been shown to regulate a wide range of physiological processes, including proliferation, differentiation, motility, cytoskeleton rearrangements, and calcium homeostasis (1, 2). There is convincing experimental evidence that this bioactive sphingolipid can act both extracellularly, as a ligand for a family of five specific G protein-coupled receptors, and inside the cells, as a second messenger (3, 4). In most cell types described so far, S1P and its metabolic precursor ceramide exert antagonistic effects on cell survival with S1P being generally regarded as a survival signal, whereas ceramide and sphingosine are generally toxic (5, 6). Interestingly, generation of sphingosine and S1P is generally thought to be dependent on the availability of ceramide (7), however, relatively high amounts of S1P are also present in blood, lymph, and cerebrospinal fluid (8, 9) and may serve as additional sources for some cells.More than a decade ago, we introduced the synthetic sphingosine analog cis-4-methylsphingosine as a tool for studies of sphingoid base metabolism and function (10). When added to the culture medium, this analog is taken up and mainly phosphorylated to the respective cis-4-methylsphingosine phosphate, which accumulates intracellularly, because it is poorly cleaved (if at all) by S1P lyase (10). Intriguingly, this compound promoted proliferation of quiescent Swiss 3T3 fibroblasts (11), as does S1P (12), but induced apoptosis in postmitotic terminally differentiated primary cultured neurons (13).Despite the fact that neither S1P nor sphingosine were able to induce apoptosis in neurons, we proposed that cis-4-methylsphingosine is phosphorylated by cells yielding a metabolically stable analog of S1P. This prediction was based on experimental results indicating that the different physiological effects, apoptosis in the case of the accumulating metabolically stable synthetic compound versus no apoptosis in the case of the short living S1P, rely only on nuances of impact (13). Both sphingoid phosphates affected similar pathways. However, the effect of the synthetic accumulated compound was more pronounced and persistent when compared with the more transient and less pronounced effect of the short living physiological counterpart (13). We therefore assumed that conditions that allow sufficient accumulation of S1P in primary cultured neurons should end up in neuronal apoptosis.To explore this hypothesis, which might be relevant to neurodegenerative processes, we attempted to elevate intracellular S1P using siRNAs directed to S1P lyase (encoded by the Sgpl1 gene). However, suppression of lyase by ∼70% did not result in an accumulation of endogenous S1P in primary cultured neurons (14).The central aim of the present study was to evaluate the hypothesis that endogenous S1P induces neuronal apoptosis when it exceeds a certain threshold by a more effective method for lyase activity suppression. We thus used primary cultured neurons prepared from cerebella of 6-day-old lyase-deficient mice (15). The present studies not only confirmed that elevation of S1P induced cell death but also revealed that the origin of the S1P was important. Intriguingly, neuronal apoptosis was induced only by S1P derived from exogenous S1P that was dephosphorylated and then resynthesized to S1P by sphingosine kinase 2 (SK2). Interestingly, we then found that this is also the kinase responsible for synthesis of cis-4-methylsphingosine phosphate. In addition, our data document that the pro-apoptotic effect of S1P is independent of cellular ceramide content.     

Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH₂ terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1, 2). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3–7). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH₂-terminal Ca²⁺-binding protein of the S-100 family, linked to 10–20 tandem filaggrin monomer repeats (8–10). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2, 11–13). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(γ-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4, 6, 14, 15). Deimination is catalyzed by peptidylarginine deiminase (PAD),² which converts arginine to citrulline in proteins (17–19). The modification seems essential for the processing into free amino acids including citrulline.Several proteases reportedly participate in the processing of profilaggrin. Furin, a member of the proprotein convertase family, has been proposed to cleave the NH₂ terminus of profilaggrin, facilitating the release of the NH₂-terminal S-100 protein (20, 21). In contrast, calpain I and profilaggrin endopeptidase I (PEP-I) were implicated in the processing of the linker regions between the filaggrin monomer repeats to generate the filaggrin monomer (22–25). Recently, significant results regarding the conversion of profilaggrin to filaggrin have been obtained with the knock-out of matriptase/MT-SP1, prostasin/channel-activating serine protease 1/Prss 8, and caspase 14 in mice (26–28). These proteases were a key component of the profilaggrin-processing pathway in terminal epidermal differentiation. However, although the signal initiating the degradation of profilaggrin at a defined stage of the maturation of the stratum corneum was found to be the water gradient within the stratum corneum itself (11), the proteases for the processing of filaggrin and/or the deiminated form into peptides following the breakdown of these peptides to amino acids including citrulline remain unknown.In this study, we have purified a novel aminopeptidase using a deiminated substrate from rat skin homogenate and identified it as a neutral cysteine protease, bleomycin hydrolase (BH). Furthermore, we investigated the processing of the deiminated filaggrin by calpain I or caspase 14. Based on these results, we proposed that calpain I participated preferentially in the processing of deiminated filaggrin into peptides and then BH appeared essential for the breakdown of the peptides into amino acids.     

Domain Interplay Mediated by an Essential Disulfide Linkage Is Critical for the Activity and Secretion of the Metastasis-promoting Enzyme Autotaxin

Autotaxin or NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2) is a secreted lysophospholipase-D that promotes metastasis and tumor growth by its ability to generate lysophosphatidic acid. Considerable evidence suggests that inhibitors of NPP2 can be used as a novel therapy for the treatment of cancer. Although most attention is currently directed toward the development of inhibitors of the catalytic site, we have explored whether NPP2 can also be targeted through its non-catalytic nuclease-like domain. We demonstrate here that the catalytic and nuclease-like domains are covalently linked by an essential disulfide bridge between Cys⁴¹³ and Cys⁸⁰⁵. Within the nuclease-like domain, residues 829–850 are involved in the secretion of NPP2, and Lys⁸⁵² is required for the expression of catalytic activity. These data show that the nuclease-like domain is crucial for catalysis by NPP2 and is a possible target to generate inhibitors.NPP2 ³ or autotaxin is a secreted lysophospholipase-D that acts in a paracrine or autocrine manner. The major substrate of NPP2 is lysophosphatidylcholine, which is converted into choline and lysophosphatidic acid. The latter promotes signaling through specific G-protein-coupled receptors that stimulate cell proliferation, differentiation, and motility (1). NPP2 functions in processes as diverse as the homing of lymphocytes, blood vessel formation, and wound healing but also promotes tumorigenesis (2–6). The metastasis-enhancing properties of NPP2 have been attributed to its ability to promote the invasive properties of cancer cells and to stimulate angiogenesis. Importantly, NPP2 is highly expressed by various cancers including breast carcinoma (7), Hodgkin lymphoma (8), and glioblastoma multiforme (9), and this correlates with an increased metastasis and angiogenesis. Therefore, NPP2 is considered to be an attractive target for a novel anticancer therapy, in particular because it acts extracellularly, and interfering drugs thus do not need to be cell-permeable (10).NPP2 is one of the seven mammalian members of the NPP-type family of ectophosphodiesterases, belonging to the superfamily of phospho-/sulfo-coordinating metalloenzymes (11). These enzymes all have a structurally related catalytic domain and the same catalytic mechanism but show a different substrate specificity. For example, NPP1 only recognizes nucleotide substrates, whereas NPP2 preferentially hydrolyzes lysophospholipids. Interestingly, NPP1–3 share a C-terminal non-catalytic domain, commonly referred to as the nuclease-like domain (NLD) (12). The latter is related to DNA/RNA-nonspecific endonucleases but lacks key residues that are necessary for activity and is therefore believed to have a structural or regulatory role. It is not known whether the NLD acts as a positive or negative regulator of NPP activity, but swapping experiments of the NLD between NPP1 and NPP2 suggested that it harbors isoform-specific determinants for catalysis (13).To examine the therapeutic potential of NPP2, in particular for the treatment of cancer, a number of small molecule inhibitors have been developed (14, 15). However, these inhibitors are all directed against the catalytic site and may therefore also interfere with other phospho-/sulfo-coordinating metalloenzymes. We report here that the NLD is essential for the expression of catalytic activity and can be used as an alternative target to inhibit NPP2.