Nucleoside diphosphate kinases (NDPKs) are encoded by nme genes and exist in various isoforms. Based on interactions with other proteins, they are involved in signal transduction, development and pathological processes such as tumorigenesis, metastasis and heart failure. In this study, we report a 1.25 Å resolution structure of human homohexameric NDPK-C bound to ADP and describe the yet unknown complexes formed with GDP, UDP and cAMP, all obtained at a high resolution via X-ray crystallography. Each nucleotide represents a distinct group of mono- or diphosphate purine or pyrimidine bases. We analyzed different NDPK-C nucleotide complexes in the presence and absence of Mg2+ and explain how this ion plays an essential role in NDPKs’ phosphotransferase activity. By analyzing a nucleotide-depleted NDPK-C structure, we detected conformational changes upon substrate binding and identify flexible regions in the substrate binding site. A comparison of NDPK-C with other human isoforms revealed a strong similarity in the overall composition with regard to the 3D structure, but significant differences in the charge and hydrophobicity of the isoforms’ surfaces. This may play a role in isoform-specific NDPK interactions with ligands and/or important complex partners like other NDPK isoforms, as well as monomeric and heterotrimeric G proteins. Considering the recently discovered role of NDPK-C in different pathologies, these high-resolution structures thus might provide a basis for interaction studies with other proteins or small ligands, like activators or inhibitors.

Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.

Plant gamma-glutamylcysteine ligase (GCL), catalyzing the first and tightly regulated step of glutathione (GSH) biosynthesis, is redox-activated via formation of an intramolecular disulfide bond. In vitro, redox-activation of recombinant GCL protein causes formation of homo-dimers. Here, we have investigated whether dimerization occurs in vivo and if so whether it contributes to redox-activation. FPLC analysis indicated that recombinant redox-activated WT (wild type) AtGCL dissociates into monomers at concentrations below 10(-6) M, i.e. below the endogenous AtGCL concentration in plastids, which was estimated to be in the micromolar range. Thus, dimerization of redox-activated GCL is expected to occur in vivo. To determine the possible impact of dimerization on redox-activation, AtGCL mutants were generated in which salt bridges or hydrophobic interactions at the dimer interface were interrupted. WT AtGCL and mutant proteins were analyzed by non-reducing SDS-PAGE to address their redox state and probed by FPLC for dimerization status. Furthermore, their substrate kinetics (K-M, V-max) were compared. The results indicate that dimer formation is not required for redox-mediated enzyme activation. Also, crystal structure analysis confirmed that dimer formation does not affect binding of GSH as competitive inhibitor. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated.

About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs.

Ras-specific GTPase-activating proteins (RasGAPs) down-regulate the biological activity of Ras proteins by accelerating their intrinsic rate of GTP hydrolysis, basically by a transition state stabilizing mechanism. Oncogenic Ras is commonly not sensitive to RasGAPs caused by interference of mutants with the electronic or steric requirements of the transition state, resulting in up-regulation of activated Ras in respective cells. RasGAPs are modular proteins containing a helical catalytic RasGAP module surrounded by smaller domains that are frequently involved in the subcellular localization or contributing to regulatory features of their host proteins. In this review, we summarize current knowledge about RasGAP structure, mechanism, regulation, and dual-substrate specificity and discuss in some detail neurofibromin, one of the most important negative Ras regulators in cellular growth control and neuronal function.

In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content ( NF1 +/−, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro , indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.

Whereas enzymes in the fumarylacetoacetate hydrolase (FAH) superfamily catalyze several distinct chemical reactions, the structural basis for their multi-functionality remains elusive. As a well-studied example, human FAH domain-containing protein 1 (FAHD1) is a mitochondrial protein displaying both acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. As mitochondrial ODx, FAHD1 acts antagonistically to pyruvate carboxylase, a key metabolic enzyme. Despite its importance for mitochondrial function, very little is known about the catalytic mechanisms underlying FAHD1 enzymatic activities, and the architecture of its ligated active site is currently ill defined. We present crystallographic data of human FAHD1 that provide new insights into the structure of the catalytic center at high resolution, featuring a flexible ‘lid’-like helical region which folds into a helical structure upon binding of the ODx inhibitor oxalate. The oxalate-driven structural transition results in the generation of a potential catalytic triad consisting of E33, H30 and an associated water molecule. In silico docking studies indicate that the substrate is further stabilized by a complex hydrogen-bond network, involving amino acids Q109 and K123, identified herein as potential key residues for FAHD1 catalytic activity. Mutation of amino acids H30, E33 and K123 each had discernible influence on the ApH and/or ODx activity of FAHD1, suggesting distinct catalytic mechanisms for both activities. The structural analysis presented here provides a defined structural map of the active site of FAHD1 and contributes to a better understanding of the FAH superfamily of enzymes.

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1–related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.

The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as "Ragulator," controls the activity of mTOR complex 1 (mTORC1) on the lysosome. The crystal structure of LAMTOR consists of two roadblock/LC7 domain-folded heterodimers wrapped and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through their carboxyl-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site-directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components to ensure fidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino acid regions in recruitment and stabilization of the Rag GTPases.

Neurofibromin and Sprouty-related EVH1 domain-containing protein 1 (Spred1) both act as negative regulators of the mitogen-activated protein kinase pathway and are associated with the rare diseases Neurofibromatosis type 1 and Legius syndrome, respectively. Spred1 recruits the major GTPase activating protein (GAP) neurofibromin from the cytosol to the membrane in order to inactivate the small G protein Ras. These functions are dependent on the N-terminal EVH1 domain and the C-terminal Sprouty domain of Spred1 whereas the former specifically recognizes the GAP related domain of neurofibromin and the latter is responsible for membrane targeting. Within the GAP domain, Spred1 binding depends on the GAPex portion which is dispensable for Ras inactivation. In a first step towards the characterization of the Neurofibromin Spred1 interface in solution we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of the Spred1 derived EVH1 domain. Our chemical shift data analysis indicate seven consecutive β-strands followed by a C-terminal α-helix which is in agreement with the previously reported crystal structure of Spred1(EVH1). Our data provide a framework for further analysis of the function of patient-derived mutations associated with rare diseases.

Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of neurofibromin that is present in all known canonical RasGAPs.

The monoclonal antibody N14 is used as a detection antibody in ELISA kits for the human glycoprotein afamin, a member of the albumin family, which has recently gained interest in the capture and stabilization of Wnt signalling proteins, and for its role in metabolic syndrome and papillary thyroid carcinoma. As a rare occurrence, the N14 Fab is N-glycosylated at Asn26L at the onset of the V L 1 antigen-binding loop, with the α-1–6 core fucosylated complex glycan facing out of the L1 complementarity-determining region. The crystal structures of two non-apparent (pseudo) isomorphous crystals of the N14 Fab were analyzed, which differ significantly in the elbow angles, thereby cautioning against the overinterpretation of domain movements upon antigen binding. In addition, the map quality at 1.9 Å resolution was sufficient to crystallographically re-sequence the variable V L and V H domains and to detect discrepancies in the hybridoma-derived sequence. Finally, a conservatively refined parsimonious model is presented and its statistics are compared with those from a less conservatively built model that has been modelled more enthusiastically. Improvements to the PDB validation reports affecting ligands, clashscore and buried surface calculations are suggested.

The protease in the commonly used commercial low-foam enzyme cleaner Zymit cannot be completely blocked by EDTA, a widely used inhibitor of metalloproteases, at concentrations of up to 5 m M . Severe protein degradation was observed in crystallization drops after EDTA-containing wash steps unless residual Zymit protease was removed with NaOH at a concentration of at least 0.1 M . Wash steps with 0.1% SDS were also ineffective in completely removing the remaining Zymit activity. Protocols including wash steps with at least 0.1 M NaOH, as for example specified in the original ZENM protocol, are recommended to completely deactivate Zymit protease activity.

Protein glycosylation plays an important role in protein stability, folding, and secretion but presents a major challenge for protein crystallization. Crystallization of highly glycosylated proteins is often difficult because of conformational and chemical inhomogeneity of the glycan decorations. Therefore, it is almost always necessary to modify the material to obtain a conformational homogenous protein that can crystallize. (1) We have explored several avenues in the case of the human 87 kDa glycoprotein afamin. Afamin (AFM), a member of the albumin gene family, is mainly expressed in the liver and secreted into the bloodstream. (2) Elevated afamin plasma concentrations are associated with major diseases such as metabolic syndrome and cancer;(3) however, pathophysiological functions are largely unknown. Therefore, we are pursuing the crystal structure of various forms of recombinantly expressed human afamin (rhAFM) for structure guided exploration and analysis of its function. Multiple variants of rhAFM are pursued: 1) fully glycosylated wild-type rhAFM (expressed in CHO cell lines); 2) rhAFM complexed with Fab fragments of two anti-AFM mAbs, 3) enzymatic deglycosylation of rhAFM with PGNaseF, 4) partially glycosylated rhAFM (expressed in glycosylation-deficient Lec1-CHO cells); and 5) glycosylation-free rhAFM, obtained from HEK293 cells virally transfected with a cDNA mutant lacking all 5 glycosylation sites by replacement of ASN with ASP. Yields of up to 2 mg/mL glycosilation free mutant rhAFM in HEK273 cells have been comparable to native rhAFM. C-terminal His6-tagged rhAFM was captured by Ni-IMAC from serum-free cell culture supernatants. All variants and Fab-AFM complexes were polished via SEC yielding pure products per SDS-PAGE and immunoblot analysis. Crystals have been obtained from Fab fragments and crystalline spherulites which are useful for microseeding from Fab-AFM complexes.

(Truman et al., 1994). Our results show an unexpected MHCII overexpressing phenotype of NF1-deficient SCs anticipating further in depth analysis as it has several potential important implications. The phenotype may have a role in the pathogenesis of neurofibroma through tumor and immune cell interactions. It would be interesting to search for tumor-associated antigens which may be presented on neurofibroma cells and could serve as therapeutic targets. HLA-DR expression itself which induces pro-apoptotic and anti-proliferative signals after ligation may be useful for therapeutic intervention. Given the abundance of MHCII expression in neurofibromin-deficient SCs targeting of these tumorigenic cells via cytotoxinconjugated antibodies is also an option for future therapeutic strategies. Furthermore, it will be interesting to evaluate the potential benefit of a combinatorial approach of anti-HLA-DR antibodies with other compounds that have been shown to be pro-apoptotic and antiproliferative in NF1-deficient cells such as MEK inhibitors (See et al., 2012). In any case mouse models will be necessary to further investigate this unusual phenotype.

TO THE EDITOR Neurofibromatosis type I (NF1) is one of the most common autosomal dominant inherited diseases affecting 1 in 3,500 individuals. It is caused by a germline mutation in the tumor suppressor gene NF1 (Viskochil et al., 1990Viskochil D. Buchberg A.M. Xu G. et al.Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus.Cell. 1990; 62: 187-192Abstract Full Text PDF PubMed Scopus (896) Google Scholar; Wallace et al., 1990Wallace M.R. Marchuk D.A. Andersen L.B. et al.Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients.Science. 1990; 249: 181-186Crossref PubMed Scopus (1211) Google Scholar). Hallmarks are the development of café au lait spots and dermal as well as plexiform neurofibromas (Riccardi, 1999Riccardi V.M. Neurofibromatosis: Phenotype, Natural History and Pathogenesis. The John Hopkins University Press, Baltimore1999Google Scholar). Neurofibromas are heterogeneous tumors composed of Schwann cells (SCs) in addition to fibroblasts, perineurial cells, mast cells, and lymphocytes (Serra et al., 2000Serra E. Rosenbaum T. Winner U. et al.Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations.Hum Mol Genet. 2000; 9: 3055-3064Crossref PubMed Scopus (195) Google Scholar). An additional somatic mutation in the second copy of the NF1 gene in a SC population is an essential early step for neurofibroma formation (Zhu et al., 2002Zhu Y. Ghosh P. Charnay P. et al.Neurofibromas in NF1: Schwann cell origin and role of tumor environment.Science. 2002; 296: 920-922Crossref PubMed Scopus (494) Google Scholar). In neurofibroma tissues both cells with biallelic NF1 inactivation (-/-) and those still bearing an intact copy of the NF1 gene (+/-) are present (Serra et al., 2000Serra E. Rosenbaum T. Winner U. et al.Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations.Hum Mol Genet. 2000; 9: 3055-3064Crossref PubMed Scopus (195) Google Scholar). In a suppressive subtractive hybridization screen comparing mRNA pools of human neurofibroma derived +/- and -/- SCs, we identified the major histocompatibility complex (MHC) class II family to be differentially expressed. MHCII genes are normally highly expressed selectively on professional antigen presenting immune cells (Krawczyk and Reith, 2006Krawczyk M. Reith W. Regulation of MHC class II expression, a unique regulatory system identified by the study of a primary immunodeficiency disease.Tissue antigen. 2006; 67: 183-197Crossref PubMed Scopus (52) Google Scholar). However, expression has also been detected in several solid tumors such as malignant melanoma and glioma (Tran et al., 1998Tran C.T. Wolz P. Egensperger R. et al.Differential expression of MHC class II molecules by microglia and neoplastic astroglia: relevance for the escape of astrocytoma cells from immune surveillance.Neuropathol Appl Neurobiol. 1998; 24: 293-301Crossref PubMed Scopus (73) Google Scholar; Campoli and Ferrone, 2008Campoli M. Ferrone S. HLA antigen changes in malignant cells: epigenetic mechanisms and biologic significance.Oncogene. 2008; 27: 5869-5885Crossref PubMed Scopus (294) Google Scholar). We generated highly enriched cultures of NF1+/- and NF1-/- SCs from neurofibromas, as well as NF1+/+ normal human SCs from peripheral nerves. NF1+/- and NF1-/- SCs were enriched from the same neurofibroma tissue derived from patients with NF1. NF1 genotype was monitored using loss of heterozygosity analysis. In addition western blot analysis for the NF1 gene product neurofibromin was performed. All cultures were set on the same culture medium for at least 3 days before expression analysis were performed (see Supplementary Methods). In western blot analysis MHCII (HLA-DR, DQ, and DP) expression was strong in all neurofibromin-deficient NF1-/- SC cultures (10/10) and barely or not detectable in cultures from NF1+/- (8/8) and NF1+/+ SCs (2/2) (Figure 1a). Neurofibroma-derived fibroblasts and NF1-/- murine embryonal fibroblasts did not express MHCII (data not shown), indicating cell type specificity of the MHCII expressing phenotype. We examined 20 neurofibromas (15 dermal and 5 plexiform) of unrelated patients for HLA-DR, DQ, and DP expression by immunohistochemistry. We observed a consistent pattern of expression with a subpopulation of MHCII-positive spindle cells lying side by side with negative ones (Figure 1b). To further characterize the MHCII-positive cells, we used double and triple immunofluorescence labeling. S100B served as marker for SCs. Using triple immunofluorescence labeling, we could show that MHCII expression is mainly restricted to S100B-positive and neurofibromin-negative SCs, whereby neurofibromin-positive SCs stained negative for MHCII (Figure 1c). MHCII expression was also detectable in lysates from neurofibroma tissues including dermal and plexiform variants. RT-PCR analysis of cultured NF1-/- SCs showed expression of all MHC class II members suggesting involvement of the class II transactivator (CIITA), known as the master regulator of MHC class II gene expression (Krawczyk and Reith, 2006Krawczyk M. Reith W. Regulation of MHC class II expression, a unique regulatory system identified by the study of a primary immunodeficiency disease.Tissue antigen. 2006; 67: 183-197Crossref PubMed Scopus (52) Google Scholar). Consistent with the dominant role of CIITA in the regulation of these genes, we found CIITA mRNA and HLA-DRα mRNA and protein levels strongly upregulated in NF1-/- SCs (Figure 1d–e). Targeting CIITA transcripts with small interfering RNA (siRNA) results in the downregulation of MHCII protein levels, demonstrating CIITA dependence of MHCII expression in NF1-/- SCs (Figure 1f). MHC class II molecules are best known for their role in antigen presentation. However, there is evidence from lymphoid as well as from melanoma cells that HLA-DR molecules are capable of transducing intracellular signals after ligand or antibody binding (Altomonte et al., 1999Altomonte M. Pucillo C. Maio M. The overlooked "nonclassical" functions of major histocompatibility complex (MHC) class II antigens in immune and nonimmune cells.J Cell Physiol. 1999; 179: 251-256Crossref PubMed Scopus (20) Google Scholar). Anti-HLA-DR antibodies have been shown to exert anti-proliferative and pro-apoptotic activity on several hematological neoplasias in vitro and in vivo (Nagy et al., 2002Nagy Z.A. Hubner B. Lohning C. et al.Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells.Nat Med. 2002; 8: 801-807Crossref PubMed Scopus (132) Google Scholar). Although an isotype-matched anti-MHCI (HLA-A,B,C) or non targeting isotype control antibody failed to exert a significant anti-proliferative effect, anti-MHCII (HLA-DR) antibody L243 reduced the growth of three different NF1-/- SC cultures significantly. Normal human SCs with low-level HLA-DR expression did not show reduced cell numbers upon L243 treatment (Figure 2a). Shortening the assay time from 72 to 24hours increased the inhibitory effect of L243 toward NF1-/- SCs but not toward normal human SCs, suggesting a rapid and transient effect (Figure 2b). Already after 5hours of incubation of NF1-/- SCs with L243 the number of living cells decreased by 20%±3 suggesting the occurrence of cell death. Terminal transferase dUTP nick-end labeling assays performed after 2.5hours of treatment confirmed the rapid induction of apoptotic cell death in L243-treated NF1-/- SCs (Figure 2c). Furthermore, cell cycle analysis showed a significant increase of the G2/G1 ratio in L243-treated cells indicative of a G2 arrest (Figure 2d). siRNA-mediated knockdown of CIITA itself did not lead to significant changes in apoptotic rate or cell cycle distribution but protects neurofibromin-deficient SCs from anti-HLA-DR antibody-induced cell death and G2 arrest (Figure 2e). Of note, the sensitivity for L243 treatment decreased in higher passage cells with lower proliferation rate, suggesting that sensitivity depends on the activation status of the cells as shown for lymphoid cells before (Truman et al., 1994Truman J.P. Ericson M.L. Choqueux-Seebold C.J. et al.Lymphocyte programmed cell death is mediated via HLA class II DR.Int Immunol. 1994; 6: 887-896Crossref PubMed Scopus (99) Google Scholar). Our results show an unexpected MHCII overexpressing phenotype of NF1-deficient SCs anticipating further in depth analysis as it has several potential important implications. The phenotype may have a role in the pathogenesis of neurofibroma through tumor and immune cell interactions. It would be interesting to search for tumor-associated antigens which may be presented on neurofibroma cells and could serve as therapeutic targets. HLA-DR expression itself which induces pro-apoptotic and anti-proliferative signals after ligation may be useful for therapeutic intervention. Given the abundance of MHCII expression in neurofibromin-deficient SCs targeting of these tumorigenic cells via cytotoxin-conjugated antibodies is also an option for future therapeutic strategies. Furthermore, it will be interesting to evaluate the potential benefit of a combinatorial approach of anti-HLA-DR antibodies with other compounds that have been shown to be pro-apoptotic and anti-proliferative in NF1-deficient cells such as MEK inhibitors (See et al., 2012See W.L. Tan I.L. Mukherjee J. et al.Sensitivity of glioblastomas to clinically available MEK inhibitors is defined by neurofibromin 1 deficiency.Cancer Res. 2012; 72: 3350-3359Crossref PubMed Scopus (76) Google Scholar). In any case mouse models will be necessary to further investigate this unusual phenotype. This work was supported by Deutsche Krebshilfe (grant 105717) and BMBF (grant 01GM0841). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid Download .pdf (.08 MB) Help with pdf files Supplementary Information

The initial reports on pleckstrin homology (PH) domains almost 20 years ago described them as sequence feature of proteins involved in signal transduction processes. Investigated at first along the phospholipid binding properties of a small subset of PH representatives, the PH fold turned out to appear as mediator of phosphotyrosine and polyproline peptide binding to other signaling proteins. While phospholipid binding now seems rather the exception among PH‐like domains, protein–protein interactions established as more and more important feature of these modules. In this review we focus on the PH superfold as a versatile protein–protein interaction platform and its three‐dimensional integration in an increasing number of available multidomain structures.