The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues.

Plant hormones are vital compounds that function as signaling molecules and control a wide range of physiological responses so that plants can cope with fluctuating environments (1). Often, plant hormones induce physiological responses locally (2–6); thus, the endogenous levels as well as the signal transduction pathways of plant hormones are regulated very precisely. Endogenous concentrations of bioactive plant hormones are primarily determined by the balance between the rates of biosynthesis and degradation (or inactivation). Additionally, specific transport systems for plant hormones and their related compounds are also considered to be critical components for generating appropriate hormone concentrations within cells, since hormone biosynthesis and perception do not necessarily take place in the same cell. In fact, movement of plant hormones and their precursors has been reported (7).Auxin is the best-characterized mobile plant hormone that plays crucial roles in many aspects of plant growth and development by mainly regulating cell division, cell elongation, and cell differentiation (8). Indole-3-acetic acid (IAA) is the major naturally occurring auxin. Measurements of IAA by mass spectrometry as well as visualization of auxin responses by reporter genes have suggested that IAA is differentially distributed within plant tissues (9). Furthermore, appropriate IAA gradients or local IAA maxima/minima are required for proper auxin functions (9). In fact, mutants impaired in IAA transport have been isolated based on their defects in organ development and various physiological responses (9). Evidence for IAA transport in a cell-to-cell manner by combinations of several transmembrane transporters is well documented. The plant-specific PIN-FORMED (PIN) transporters and the AUX1/LAX family of amino acid permease-like proteins are IAA efflux and influx carriers, respectively (10, 11). PINs determine the direction of IAA flow by localizing unevenly in the plasma membrane, whereas polarity is not observed for AUX/LAXs distribution (9). Also, some subfamily B members of the adenosine 5′-triphosphate-binding cassette (ABC) transporter (ABCBs) family act as IAA transporters (9).IAA is mainly synthesized from tryptophan via indole-3-pyruvic acid (12); however, minor routes for IAA biosynthesis have also been suggested (13). Indole-3-butyric acid (IBA) has auxin activity when applied exogenously to plants. Arabidopsis mutants isolated based on their resistance to exogenous IBA are defective in the conversion of IBA to IAA (14). Since IBA has been detected in several plant species (13), the compound is considered to be an endogenous IAA precursor. Possible IBA transporters have been identified (15–17), suggesting that IBA transport is also an important factor that regulates endogenous IAA levels.NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY (NPF) proteins have been characterized as transporters for several plant hormones including auxin, although the family was initially identified as nitrate or di/tripeptide transporters (18, 19). The Arabidopsis NPF6.3 protein (also called as CHL1 or NRT1.1) is a well-characterized dual-affinity nitrate transporter that mediates nitrogen uptake from the rhizosphere (20, 21); however, the same protein also transports IAA (22). More recently, the Arabidopsis NPF5.12 protein, also named TRANSPORTER OF IBA1 (TOB1), was identified as an IBA transporter that localizes to the vacuolar membrane (17). Here, we report that NPF7.3, originally identified as a low-affinity nitrate transporter (NRT1.5) and later shown to be a proton/potassium antiporter in Arabidopsis (23, 24), functions as an IBA transporter. We found that npf7.3 mutant roots were not able to respond properly to gravity. Our transport assays in yeast demonstrated that NPF7.3 efficiently mediated IBA uptake into the cells. Asymmetric distribution of auxin activities in root tips following the reorientation of roots was not observed in the npf7.3 mutants, and the defect was restored by IAA but not by IBA treatment. These results suggest that cellular IBA uptake mediated by NPF7.3 contributes to the production of IAA required to fully induce Arabidopsis root gravitropism.     

SNAP-29: A general SNARE protein that inhibits SNARE disassembly and is implicated in synaptic transmission

Using the yeast two-hybrid system with syntaxin-1A as bait, we isolated soluble NSF attachment protein (SNAP)-29 from a human brain cDNA library. Synaptosomal fractionation and immunocytochemical staining of hippocampal neurons in culture showed that SNAP-29 is present at synapses and is predominantly associated with synaptic vesicles. The interaction of SNAP-29 with syntaxin-1 was further confirmed with immunoprecipitation analysis. Binding competition studies with SNAP-29 demonstrated that it could compete with alpha-SNAP for binding to synaptic SNAP receptors (SNAREs) and consequently inhibit disassembly of the SNARE complex. Introduction of SNAP-29 into presynaptic superior cervical ganglion neurons in culture significantly inhibited synaptic transmission in an activity-dependent manner. Although SNAP-29 has been suggested to be a general SNARE component in membrane trafficking, our findings suggest that it may function as a regulator of SNARE complex disassembly and modulate the process of postfusion recycling of the SNARE components.     

Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis

MicroRNAs (miRNAs) are important for plant development and stress responses. However, factors regulating miRNA metabolism are not completely understood. SICKLE (SIC), a proline-rich protein critical for development and abiotic stress tolerance of Arabidopsis, was identified in this study. Loss-of-function sic-1 mutant plants exhibited a serrated, sickle-like leaf margin, reduced height, delayed flowering, and abnormal inflorescence phyllotaxy, which are common characteristics of mutants involved in miRNA biogenesis. The sic-1 mutant plants accumulated lower levels of a subset of miRNAs and transacting siRNAs but higher levels of corresponding primary miRNAs than the WT. The SIC protein colocalizes with the miRNA biogenesis component HYL1 in distinct subnuclear bodies. sic-1 mutant plants also accumulated higher levels of introns from hundreds of loci. In addition, sic-1 mutant plants are hypersensitive to chilling and salt stresses. These results suggest that SIC is a unique factor required for the biogenesis of some miRNAs and degradation of some spliced introns and important for plant development and abiotic stress responses.     

A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex.

An approach to identifying the interaction site of multicomponent protein assembly has been applied to the membrane-bound SecY-SecE complex, which mediates protein export across the Escherichia coli cytoplasmic membrane. A dominant negative secY allele, secY-d1, inactivates SecY but preserves its ability to interact with SecE. Thus, the mutant protein sequesters SecE in an inactive complex. Second site mutations that disrupt the SecE binding site will suppress the export interference. We introduced insertion/deletion mutations that intragenically suppressed secY-d1. After eliminating knock-out mutations by virtue of the expression of a LacZ alpha sequence that had been attached to the C terminus, we obtained a striking clustering of mutations in cytoplasmic domain 4. On the basis of this result, the secY24 (Ts) substitution mutation in this domain was examined for its effects on interaction with SecE. It indeed suppressed secY-d1. Although the instability associated with excess SecY can be alleviated by overproduction of SecE, the secY24 mutant protein was not stabilized by SecE. The basal-level SecY24 protein was also destabilized at 42 degrees C. SecE was coimmunoprecipitated with SecY+ but not with the SecY24 protein. These results indicate that the secY24 mutation weakens SecY's interaction with SecE. Taken together, we propose that cytoplasmic domain 4 is important for the association between SecY and SecE.     

Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis

RNA silencing in plants and some animals has a non–cell-autonomous effect due to an RNA signal that moves between cells or organs. To identify unique factors involved in this process, we analyzed a group of Arabidopsis mutants with defective spread of RNA silencing from a transgene expressed specifically in the phloem. These mutants accumulated reduced amounts of small interfering (si)RNA from the transgene locus and from endogenous loci TAS1, TAS2, and an inverted repeat locus IR71. The defect in TAS1 and TAS2 siRNA biogenesis is in the processing of a long siRNA precursor. We mapped the mutations to a gene encoding the Arabidopsis homolog of a protein, TEX1, which is involved in intracellular transport of RNA in animals. TEX1 is a component of the THO/TREX complex, and we show that the Arabidopsis TEX1 interacts with other predicted components of a THO/TREX complex. Correspondingly, we found at least two other components of the Arabidopsis THO core complex that are involved in RNA silencing. To reconcile the effect of these mutations on transgene and endogenous gene siRNA, we propose a mechanism in which THO/TREX processes or transports a long RNA molecule so that it can be a template for secondary siRNA production.     

A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in "cis" and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking.     

Possible roles for Munc18-1 domain 3a and Syntaxin1 N-peptide and C-terminal anchor in SNARE complex formation

Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.     

The ionic layer is required for efficient dissociation of the SNARE complex by α-SNAP and NSF

The four-helical bundle soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex that mediates intracellular membrane fusion events contains a highly conserved ionic layer at the center of an otherwise hydrophobic core. This layer has an undetermined function; it consists of glutamine (Q) residues in syntaxin and the two synaptosomal-associated protein of 25 kDa (SNAP-25) family helices, and an arginine (R) in vesicle-associated membrane protein (a 3Q:1R ratio). Here, we show that the ionic-layer glutamine of syntaxin is required for efficient alpha-SNAP and NSF-mediated dissociation of the complex. When this residue is mutated, the SNARE complex still binds to alpha-SNAP and NSF and is released through ATP hydrolysis by NSF, but the complex no longer dissociates into SNARE monomers. Thus, one function of the ionic layer--in particular, the glutamine of syntaxin--is to couple ATP hydrolysis by NSF to the dissociation of the fusion complex. We propose that alpha-SNAP and NSF drive conformational changes at the ionic layer through specific interactions with the syntaxin glutamine, resulting in the dissociation of the SNARE complex.     

A novel promoter for the 11beta-hydroxysteroid dehydrogenase type 1 gene is active in lung and is C/EBPalpha independent

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) increases intracellular glucocorticoid action by converting inactive to active glucocorticoids (cortisol, corticosterone) within cells. It is highly expressed in glucocorticoid target tissues including liver and lung, and at modest levels in adipose tissue and brain. A selective increase in adipose 11beta-HSD1 expression occurs in obese humans and rodents and is likely to be of pathogenic importance in the metabolic syndrome. Here we have used 5' rapid amplificaiton of cDNA ends (RACE) to identify a novel promoter, P1, of the gene encoding 11beta-HSD1. P1 is located 23 kb 5' to the previously described promoter, P2. Both promoters are active in liver, lung, adipose tissue, and brain. However, P1 (encoding exon 1A) predominates in lung and P2 (encoding exon 1B) predominates in liver, adipose tissue, and brain. Adipose tissue of obese leptin-deficient C57BL/6J-Lepob mice showed higher expression only of the P2-associated exon 1B-containing 11beta-HSD1 mRNA variant. In contrast to P2, which is CAAAT/enhancer binding protein (C/EBP)-alpha inducible in transiently transfected cells, the P1 promoter was unaffected by C/EBPalpha in transfected cells. Consistent with these findings, mice lacking C/EBPalpha had normal 11beta-HSD1 mRNA levels in lung but showed a dramatic reduction in levels of 11beta-HSD1 mRNA in liver and brown adipose tissue. These results therefore demonstrate tissue-specific differential regulation of 11beta-HSD1 mRNA through alternate promoter usage and suggest that increased adipose 11beta-HSD1 expression in obesity is due to a selective increase in activity of the C/EBPalpha-regulated P2 promoter.     

An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability

The Arp2/3 complex polymerizes new actin filaments from the sides of existing filaments, forming Y-branched networks that are critical for actin-mediated force generation. Binding of the Arp2/3 complex to the sides of actin filaments is therefore central to its actin-nucleating and branching activities. Although a model of the Arp2/3 complex in filament branches has been proposed based on electron microscopy, this model has not been validated using independent approaches, and the functional importance of predicted actin-binding residues has not been extensively tested. Using a combination of molecular dynamics and protein-protein docking simulations, we derived an independent structural model of the interaction between two subunits of the Arp2/3 complex that are key to actin binding, ARPC2 and ARPC4, and the side of an actin filament. This model agreed remarkably well with the previous results from electron microscopy. Complementary mutagenesis experiments revealed numerous residues in ARPC2 and ARPC4 that were required for the biochemical activity of the entire complex. Functionally critical residues clustered together and defined a surface that was predicted by protein-protein docking to be buried in the interaction with actin. Moreover, key residues at this interface were crucial for actin nucleation and Y-branching, high-affinity F-actin binding, and Y-branch stability, demonstrating that the affinity of Arp2/3 complex for F actin independently modulates branch formation and stability. Our results highlight the utility of combining computational and experimental approaches to study protein-protein interactions and provide a basis for further elucidating the role of F-actin binding in Arp2/3 complex activation and function.     

A mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase is expressed in endothelial cells and plays an important role in angiogenesis

Using polymerase chain reaction-coupled subtractive hybridization, we have isolated genes expressed during the in vitro differentiation of murine embryonic stem cells into endothelial cells (ECs). Among the genes obtained, we identified one gene that was inducible by vascular endothelial growth factor (VEGF) in the murine EC line MSS31. Analysis of the nucleotide and deduced amino acid sequences revealed that the protein was composed of 930 amino acids, including an HEXXH(X)18E consensus sequence of the M1 aminopeptidase family, and is thought to be a mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase (mPILSAP). The recombinant protein hydrolyzed N-terminal leucyl and methionyl residues from synthetic substrates. Immunohistochemical analysis revealed that mPILSAP was expressed in ECs during postnatal angiogenesis. Specific elimination of mPILSAP expression by antisense oligodeoxynucleotide (AS-ODN) attenuated VEGF-stimulated proliferation, migration, and network formation of ECs in vitro. Moreover, AS-ODN to mPILSAP inhibited angiogenesis in vivo. These results suggest a novel function of mPILSAP, which is expressed in ECs and plays an important role in angiogenesis.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes.

We have isolated the Arabidopsis thaliana gene (HMG1) encoding 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; (S)-mevalonate:NAD+ oxido-reductase (CoA-acylating), EC 1.1.1.88], the catalyst of the first committed step in isoprenoid biosynthesis. cDNA copies of the plant gene were identified by hybridization with a short, highly conserved segment of yeast HMG-CoA reductase as probe. DNA sequence analysis reveals that the COOH-terminal domain of the Arabidopsis HMG-CoA reductase (containing the catalytic site of the enzyme) is highly conserved with respect to the yeast, mammalian, and Drosophila enzymes, whereas the membrane-bound amino terminus of the Arabidopsis protein is truncated and lacks the complex membrane-spanning architecture of the yeast and animal reductases. Expression of the Arabidopsis gene from the yeast GAL1 promoter in a yeast mutant lacking HMG-CoA reductase activity suppresses the growth defect of the yeast mutant. Taken together, the sequence similarity to other cloned HMG-CoA reductase genes and the suppression of the yeast hmg- mutant provide strong evidence that the novel Arabidopsis gene we have cloned encodes a functional HMG-CoA reductase enzyme.     

A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene.     

Nucleoporin MOS7/Nup88 is required for mitosis in gametogenesis and seed development in Arabidopsis

Angiosperm reproduction is characterized by alternate diploid sporophytic and haploid gametophytic generations. Gametogenesis shares similarities with that of animals except for the formation of the gametophyte, whereby haploid cells undergo several rounds of postmeiotic mitosis to form gametes and the accessory cells required for successful reproduction. The mechanisms regulating gametophyte development in angiosperms are incompletely understood. Here, we show that the nucleoporin Nup88-homolog MOS7 (Modifier of Snc1,7) plays a crucial role in mitosis during both male and female gametophyte formation in Arabidopsis thaliana. Using a mutagenesis screen, we identify the mos7-5 mutant allele, which causes ovule and pollen abortion in MOS7/mos7-5 heterozygous plants, and preglobular stage embryonic lethality in homozygous mos7-5 seeds. During interphase, we show that MOS7 is localized to the nuclear membrane but, like many nucleoporins, is associated with the spindle apparatus during mitosis. We detect interactions between MOS7 and several nucleoporins known to control spindle dynamics, and find that in pollen from MOS7/mos7-5 heterozygotes, abortion is accompanied by a failure of spindle formation, cell fate specification, and phragmoplast activity. Most intriguingly, we show that following gamete formation by MOS7/mos7-5 heterozygous spores, inheritance of either the MOS7 or the mos7-5 allele by a given gamete does not correlate with its respective survival or abortion. Instead, we suggest a model whereby MOS7, which is highly expressed in the Pollen- and Megaspore Mother Cells, enacts a dosage-limiting effect on the gametes to enable their progression through subsequent mitoses.A most striking difference between the developmental approaches of plants and animals is the intervention of mitotic divisions between meiosis and gamete formation in plants (1). Following meiosis, successive mitotic divisions of haploid cells produce both mature gametes and complex gametophytic structures encasing them, facilitating fertilization. The regulation of gametophyte formation is not completely understood, but is characterized by elegant cell, nuclear and organelle migration, led by microtubule activity (2, 3).Arabidopsis thaliana female gametophyte formation begins with meiosis of the diploid megaspore mother cell (MMC), forming four haploid megaspores. Female Gametogenesis stages (FG) follow, with megaspores migrating to the micropylar end of the gametophyte (FG1) (4). Three megaspores degenerate, one undergoes mitosis and nuclei migrate to opposite poles, likely facilitated by development of a large central vacuole (FG2). Two additional mitotic divisions generate the eight-nuclear FG5 female gametophyte. Nuclei migrate according to their cell-fate and simultaneous cytokinesis (cellularization) takes place, followed by polar nuclei fusion to form the homo-diploid central cell (FG6). At the micropylar pole lie the synergid cells and egg, and three antipodal cells are located at the opposite pole, which degenerate, marking formation of the mature female gametophyte (FG7) (5).Male gametogenesis also involves precise nuclear migration and both symmetric and asymmetric cell divisions. Meiosis of the diploid pollen mother cell (PMC) produces a tetrad of haploid microspores (6). Unlike female megaspores, however, and reminiscent of mammalian spermatogenesis, all four microspores survive to undergo asymmetric mitotic division, Pollen Mitosis I (PMI), each producing a generative cell engulfed in the cytoplasm of a vegetative cell. The generative cell undergoes a second mitosis (PMII) to form two identical sperm cells. The vegetative nucleus gives rise to the pollen tube (7).Around half of the genes so far identified as functioning in gametogenesis regulate both female and male gametophyte formation, and are involved in common cellular processes that occur in both such as mitosis, vacuole formation, cellularization, nuclear migration and cell expansion (8). To identify genes involved in gametogenesis, we performed a mutagenesis screen for lines showing seed and ovule lethality. We identified mos7-5, a MOS7 (Modifier Of Snc1,7) mutant. MOS7 is homologous to human and Drosophila melanogaster nucleoporin protein Nup88. Nucleoporins comprise nuclear pore complexes (NPCs) which traffic proteins and RNA between the nucleus and cytoplasm (9). Previously identified mos7 mutant alleles were mos7-1, a hypomorphic loss-of-function allele that revealed the importance of MOS7-mediated nucleocytoplasmic passage of defense proteins for plant innate immunity (10); mos7-2 and mos 7–4, both embryonic lethal when homozygous (10). Thus, MOS7, as well as an immune function, likely has a developmental function that is currently unknown, although alterations in human Nup88 expression result in multipolar spindles and promote carcinogenesis (11), so MOS7 may have a role in plant cell division.     

Immunomodulation for inhibitors in hemophilia A: the important role of Treg cells

Approximately 25-30% of the hemophilia A patients develop inhibitory antibodies against Factor VIII (FVIII) following protein-replacement therapy. This problem is also thought to occur following gene-replacement therapy. Recently, many approaches have been investigated to modulate FVIII-specific immune responses in either protein-replacement or gene therapy hemophilia A mouse models. Several promising protocols have been demonstrated to successfully prevent or modulate the formation of anti-FVIII antibodies, including methods to manipulate antigen presentation, development of less immunogenic FVIII proteins, or formulations or gene therapy protocols to evade immune responses, as well as immunomodulation strategies to target either T- and/or B-cell responses. Most of these successful protocols involve the induction of activated Treg cells to create a regulatory immune environment during tolerance induction. Innovative strategies to overcome pre-existing anti-FVIII immune responses and induce long-term tolerance in primed subjects still need to be developed.