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Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis, or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global downregulation of Fe-S-containing proteins and Fe 2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53 deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids. Our results suggest that p53 status and phospholipid homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (high-molecular-weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide-MHC ligand were readily lysed by T cells transduced with genes encoding α,β-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pregnane X receptor (PXR) plays an important role in xenobiotic metabolism. While ligand binding induces PXR-dependent gene transcription, PXR shows constitutive transcriptional activity in the absence of ligands when expressed in cultured cells. This constitutive activity sometimes hampers investigation of PXR activation by compounds of interest. In this study, we investigated the molecular mechanism of PXR activation. In the reported crystal structures of unliganded PXR, helix 12 (H12), including a coactivator binding motif, was stabilized, while it is destabilized in the unliganded structures of other nuclear receptors, suggesting a role for H12 stabilization in the basal activity of PXR. Since Phe420, located in the loop between H11 and H12, is thought to interact with Leu411 and Ile414 to stabilize H12, we substituted alanine at Phe420 (PXR-F420A) and separately inserted three alanine residues directly after Phe420 (PXR-3A) and investigated their influence on PXR-mediated transcription. Reporter gene assays demonstrated that the mutants showed drastically reduced basal activity and enhanced responses to various ligands, which was further enhanced by coexpression of the coactivator peroxisome proliferator-activated receptor gamma coactivator 1α. Mutations of both Leu411 and Ile414 to alanine also suppressed basal activity. Mammalian two-hybrid assays showed that PXR-F420A and PXR-3A bound to corepressors and coactivators in the absence and presence of ligands, respectively. We conclude that the intramolecular interactions of Phe420 with Leu411 and Ile414 stabilize H12 to recruit coactivators even in the absence of ligands, contributing to the basal transcriptional activity of PXR. We propose that the generated mutants might be useful for PXR ligand screening., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state ( 15Z Pb) and orange-absorbing 15E state ( 15E Po). pH titration analysis revealed that 15Z Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, 15E Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state ( 15Z Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in 15Z Pb and bilin protonation in 15E Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated 15Z Pg and protonated 15E Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed 15Z Pg formation as an intermediate in the 15E Pr-to- 15Z Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP -to- 15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies., (© 2019 Sato et al.)

The type-I LacdiNAc (LDN; GalNAcβ1-3GlcNAc) has rarely been observed in mammalian cells except in the O -glycan of α-dystroglycan, in contrast to type-II LDN structures (GalNAcβ1-4GlcNAc) in N - and O -glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. Although a β1,3- N -acetylgalactosaminyltransferase 2 (B3GALNT2; type-I LDN synthase) has been cloned, the function of type-I LDN in mammalian cells is still unclear, as its carrier protein(s) has not been identified. In this study, using HeLa cells, we demonstrate that inhibition of Golgi-resident glycosyltransferase increases the abundance of B3GALNT2-synthesized type-I LDN structures, recognized by Wisteria floribunda agglutinin (WFA). Using isotope-coded glycosylation site-specific tagging (IGOT)-LC/MS analysis of Lec8 Chinese hamster cells lacking galactosylation and of cells transfected with the B3GALNT2 gene, we identified the glycoproteins that carry B3GALNT2-generated type-I LDN in their N -glycans. Our results further revealed that LDN presence on low-density lipoprotein receptor-related protein 1 and nicastrin depends on B3GALNT2, indicating the occurrence of type-I LDN in vivo in mammalian cells. Our analysis also uncovered that most of the identified glycoproteins localize to intracellular organelles, particularly to the endoplasmic reticulum. Whereas B4GALNT3 and B4GALNT4 synthesized LDN on extracellular glycoproteins, B3GALNT2 primarily transferred LDN to intracellular glycoproteins, thereby clearly delineating proteins that carry type-I or type-II LDNs. Taken together, our results indicate the presence of mammalian glycoproteins carrying type-I LDN on N -glycans and suggest that type-I and type-II LDNs have different roles in vivo ., (© 2019 Nakane et al.)

Some protein and peptide aggregates, such as those of amyloid-β protein (Aβ), are neurotoxic and have been implicated in several neurodegenerative diseases. Aβ accumulates at nanoclusters enriched in neuronal lipids called gangliosides in the presynaptic neuronal membrane, and the resulting oligomeric and/or fibrous forms accelerate the development of Alzheimer's disease. Although the presence of Aβ deposits at such nanoclusters is known, the mechanism of their assembly and the relationship between Aβ secondary structure and topography are still unclear. Here, we first confirmed by atomic force microscopy that Aβ 40 fibrils can be obtained by incubating seed-free Aβ 40 monomers with a membrane composed of sphingomyelin, cholesterol, and the ganglioside GM1. Using Fourier transform infrared (FTIR) reflection-absorption spectroscopy, we then found that these lipid-associated fibrils contained parallel β-sheets, whereas self-assembled Aβ 40 molecules formed antiparallel β-sheets. We also found that the fibrils obtained at GM1-rich nanoclusters were generated from turn Aβ 40 Our findings indicate that Aβ generally self-assembles into antiparallel β-structures but can also form protofibrils with parallel β-sheets by interacting with ganglioside-bound Aβ. We concluded that by promoting the formation of parallel β-sheets, highly ganglioside-enriched nanoclusters help accelerate the elongation of Aβ fibrils. These results advance our understanding of ganglioside-induced Aβ fibril formation in neuronal membranes and may help inform the development of additional therapies for Alzheimer's disease., (© 2018 Matsubara et al.)

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Disease-causing mutations in G protein-coupled receptor (GPCR) genes, including the V2 vasopressin receptor (V2R) gene, often cause misfolded receptors, leading to a defect in plasma membrane trafficking. A novel V2R mutation, T273M, identified in a boy with partial nephrogenic diabetes insipidus (NDI), shows intracellular localization and partial defects similar to the two mutants we described previously (10). Although non-peptide V2R antagonists have been shown to rescue the membrane localization of V2R mutants, their level of functional rescue is weak. Interestingly, it has been reported that a non-peptide agonist, OPC51803, activates misfolded V2R mutants intracellularly without degradation, thus potentially serving as a therapeutic agent against NDI (14). In our current experiments, however, a peptide antagonist blocked arginine vasopressin (AVP)- or OPC51803-stimulated cAMP accumulation both in COS-7 and MDCK cells, suggesting that OPC51803 mainly stimulates cell surface V2R mutants. In addition, our analyses revealed that OPC51803 works not only as a non-peptide agonist that causes activation/β-arrestin-dependent desensitization of V2R mutants expressed at the plasma membrane but also as a pharmacochaperone that promotes the endoplasmic reticulum-retained mutant maturation and trafficking to the plasma membrane. The ratio of the pharmacochaperone effect to the desensitization effect likely correlates negatively with the residual function of the tested mutants, suggesting that OPC5 has a more favorable effect on the V2R mutants with a less residual function. We speculated that the canceling of the desensitization effect of OPC51803 by the pharmacochaperone effect after long-term treatment may produce sustainable signaling, and thus pharmacochaperone agonists such as OPC51803 may serve as promising therapeutics for NDI caused by misfolded V2R mutants., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His(499) near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His(499) is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His(499) regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Regulation of spatiotemporal gene expression in higher eukaryotic cells is critical for the precise and orderly development of undifferentiated progenitors into committed cell types of the adult. It is well known that dynamic epigenomic regulation (including chromatin remodeling and histone modifications by transcriptional coregulator complexes) is involved in transcriptional regulation. Precisely how these coregulator complexes exert their cell type and developing stage-specific activity is largely unknown. In this study we aimed to isolate the histone demethylase lysine-specific demethylase 1 (LSD1) complex from neural cells by biochemical purification. In so doing, we identified myelin transcription factor 1 (MyT1) as a novel LSD1 complex component. MyT1 is a neural cell-specific zinc finger factor, and it forms a stable multiprotein complex with LSD1 through direct interaction. Target gene analysis using microarray and ChIP assays revealed that the Pten gene was directly regulated by the LSD1-MyT1 complex. Knockdown of either LSD1 or MyT1 derepressed the expression of endogenous target genes and inhibited cell proliferation of a neuroblastoma cell line, Neuro2a. We propose that formation of tissue-specific combinations of coregulator complexes is a critical mechanism for tissue-specific transcriptional regulation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr(209), Ser(247), Ser(270), and Ser(303) as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr(209) and Ser(247) on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr(209) peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Osteoclastogenesis is controlled by osteocytes; osteocytic osteoclastogenesis regulatory molecules are largely unknown. We searched for such factors using newly developed culture methods. Our culture system mimics the three-dimensional cellular structure of bone, consisting of collagen gel-embedded osteocytic MLO-Y4 cells, stromal ST2 cells on the gel as bone lining cells, and bone marrow cells. The gel-embedded MLO-Y4 cells inhibited the osteoclastogenesis induced by 1,25(OH)2D3 without modulating receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production by ST2 cells, despite MLO-Y4 cells supported osteoclastogenesis in the absence of ST2 cells. In the bone marrow cell culture, the conditioned medium from MLO-Y4 cells decreased the capability of osteoclastic differentiation from the cells induced by macrophage colony-stimulating factor. This decreased capability was concomitant with an increase in protein kinase R mRNA expression and an inhibition of c-Fos translation. These changes were partially normalized by the simultaneous addition of an anti-interferon (IFN)-β neutralizing antibody to MLO-Y4 cell conditioned medium. To study primary osteocytes, we prepared non-osteocytic cell-free osteocyte-enriched bone fragments (OEBFs). When osteoclast precursors were induced by macrophage colony-stimulating factor in the presence of OEBFs, the generated cells exhibited a diminished capacity for osteoclastogenesis. OEBFs prepared from OPG-knock-out mice exhibited a similar effect, indicating OPG-independent inhibition. The addition of anti-IFN-β neutralizing antibody during the co-culture with OEBFs partially recovered the osteoclastogenic potential of the generated cells. The MLO-Y4 cells and OEBFs expressed IFN-β mRNA. Although osteocytic RANKL is known to be important for osteoclastogenesis, our data suggest that osteocytes also produce IFN-β as an inhibitor of osteoclastogenesis.

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.

Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.

Ribose-1,5-bisphosphate isomerase (R15Pi) is a novel enzyme recently identified as a member of an AMP metabolic pathway in archaea. The enzyme converts d-ribose 1,5-bisphosphate into ribulose 1,5-bisphosphate, providing the substrate for archaeal ribulose-1,5-bisphosphate carboxylase/oxygenases. We here report the crystal structures of R15Pi from Thermococcus kodakarensis KOD1 (Tk-R15Pi) with and without its substrate or product. Tk-R15Pi is a hexameric enzyme formed by the trimerization of dimer units. Biochemical analyses show that Tk-R15Pi only accepts the α-anomer of d-ribose 1,5-bisphosphate and that Cys(133) and Asp(202) residues are essential for ribulose 1,5-bisphosphate production. Comparison of the determined structures reveals that the unliganded and product-binding structures are in an open form, whereas the substrate-binding structure adopts a closed form, indicating domain movement upon substrate binding. The conformational change to the closed form optimizes active site configuration and also isolates the active site from the solvent, which may allow deprotonation of Cys(133) and protonation of Asp(202) to occur. The structural features of the substrate-binding form and biochemical evidence lead us to propose that the isomerase reaction proceeds via a cis-phosphoenolate intermediate.

Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR(-/-)) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR(-/-) osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR(-/-) preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V(0) subunit d2 and dendritic cell-specific transmembrane protein in LDLR(-/-) plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR(-/-) mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.

Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1(-/-) mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1(-/-) cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152-4161). Histologically, the growth plate of Csgalnact1(-/-) mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1(-/-) cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.

Langerin is categorized as a C-type lectin selectively expressed in Langerhans cells, playing roles in the first line of defense against pathogens and in Birbeck granule formation. Although these functions are thought to be exerted through glycan-binding activity of the C-type carbohydrate recognition domain, sugar-binding properties of Langerin have not been fully elucidated in relation to its biological functions. Here, we investigated the glycan-binding specificity of Langerin using comprehensive glycoconjugate microarray, quantitative frontal affinity chromatography, and conventional cell biological analyses. Langerin showed outstanding affinity to galactose-6-sulfated oligosaccharides, including keratan sulfate, while it preserved binding activity to mannose, as a common feature of the C-type lectins with an EPN motif. By a mutagenesis study, Lys-299 and Lys-313 were found to form extended binding sites for sulfated glycans. Consistent with the former observation, the sulfated Langerin ligands were found to be expressed in brain and spleen, where the transcript of keratan sulfate 6-O-sulfotransferase is expressed. Moreover, such sulfated ligands were up-regulated in glioblastoma relative to normal brain tissues, and Langerin-expressing cells were localized in malignant brain tissues. Langerin also recognized pathogenic fungi, such as Candida and Malassezia, expressing heavily mannosylated glycans. These observations provide strong evidence that Langerin mediates diverse functions on Langerhans cells through dual recognition of sulfated as well as mannosylated glycans by its uniquely evolved C-type carbohydrate-recognition domain.

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G(2) and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.

beta-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the beta-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal beta-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean beta-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.

Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1 approximately 5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis.

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.

Amyloid fibril formation of mutant transthyretin (TTR) that causes familial amyloid polyneuropathy occurs in the extracellular space. Thus, secretion of TTR variants contributes to the pathogenesis of amyloidosis. However, the molecular mechanisms underlying the endoplasmic reticulum (ER) exit or retention and subsequent degradation of TTR variants remain unclear. Here, we demonstrated that the nonsecreted TTR variants, such as D18G TTR and amyloidogenic TTRs with introduced monomeric mutation (M-TTRs), stably interact with the ER chaperone BiP in mammalian cells. These proteins were co-secreted with the secreted form of BiP in which the KDEL signal was removed, indicating that BiP partially contributes to the ER retention of nonsecreted TTR variants. More interestingly, the degradation efficiency of nonsecreted TTRs was increased when BiP was down-regulated by small interfering RNA. Thus, BiP protects the TTR variants from immediate degradation. Additionally, we showed that the stability of nonsecreted TTR variants is not disturbed in the coat complex II-deficient conditions, which are enough to inhibit the ER export of secreted TTR variants, including wild-type TTR. Therefore, the post-ER retrieval mechanism might not contribute to the ER-associated degradation of nonsecreted TTR variants. These findings suggest that the affinity to the ER-resident protein BiP regulates the fate of TTR variants in the ER.

Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.

Mammalian glycan chain elongation is mostly based on extending the type 2 chain, Galbeta1-4GlcNAc, whereas the corresponding type 1 chain, Galbeta1-3GlcNAc, is not normally extended. In a broader context of developing high sensitivity mass spectrometry methodologies for glycomic identification of Le(a) versus Le(x) and linear versus branched poly-N-acetyllactosamine (polyLacNAc), we have now shown that the dimeric type 1 glycan chain, as carried on the lactosylceramides of a human colonic adenocarcinoma cell line, Colo205, not only can be further extended linearly but can likewise be branched at C6 of 3-linked Gal in a manner similar to polyLacNAc. A combination of chemical and enzymatic derivatization coupled with advanced mass spectrometry analyses afforded unambiguous identification of a complex mixture of type 1 and 2 hybrids as well as those fucosylated variants founded exclusively on linear and branched trimeric type 1 chain. We further showed by in vitro enzymatic synthesis that extended type 1 and the hybrid chains can be branched by all three forms of the human I branching enzymes (IGnT) currently identified but with lower efficiency and stringency with respect to branching site preference. Importantly, it was found that a better substrate is one that carries a Gal site for branching that is extended at the non-reducing end by a type 2 and not a type 1 unit, whereas the IGnTs are less discriminative with respect to whether the targeted Gal site is itself beta3- or beta4-linked to GlcNAc at the reducing end.

The beta-amyloid peptide (Abeta) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer disease. Abeta is produced by the sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. Cleavage of APP by gamma-secretase also generates the APP intracellular C-terminal domain (AICD) peptide, which might be involved in regulation of gene transcription. APP contains three Gly-XXX-Gly (GXXXG) motifs in its juxtamembrane and transmembrane (TM) regions. Such motifs are known to promote dimerization via close apposition of TM sequences. We demonstrate that pairwise replacement of glycines by leucines or isoleucines, but not alanines, in a GXXXG motif led to a drastic reduction of Abeta40 and Abeta42 secretion. beta-Cleavage of mutant APP was not inhibited, and reduction of Abeta secretion resulted from inhibition of gamma-cleavage. It was anticipated that decreased gamma-cleavage of mutant APP would result from inhibition of its dimerization. Surprisingly, mutations of the GXXXG motif actually enhanced dimerization of the APP C-terminal fragments, possibly via a different TM alpha-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production.

DyP, a unique dye-decolorizing enzyme from the fungus Thanatephorus cucumeris Dec 1, has been classified as a peroxidase but lacks homology to almost all other known plant peroxidases. The primary structure of DyP shows moderate sequence homology to only two known proteins: the peroxide-dependent phenol oxidase, TAP, and the hypothetical peroxidase, cpop21. Here, we show the first crystal structure of DyP and reveal that this protein has a unique tertiary structure with a distal heme region that differs from that of most other peroxidases. DyP lacks an important histidine residue known to assist in the formation of a Fe4+ oxoferryl center and a porphyrin-based cation radical intermediate (compound I) during the action of ubiquitous peroxidases. Instead, our tertiary structural and spectrophotometric analyses of DyP suggest that an aspartic acid and an arginine are involved in the formation of compound I. Sequence analysis reveals that the important aspartic acid and arginine mentioned above and histidine of the heme ligand are conserved among DyP, TAP, and cpop21, and structural and phylogenetic analyses confirmed that these three enzymes do not belong to any other families of peroxidase. These findings, which strongly suggest that DyP is a representative heme peroxidase from a novel family, should facilitate the identification of additional new family members and accelerate the classification of this novel peroxidase family.

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.

Malignant transformation is associated with increased gene expression of beta-1,4-galactosyltransferase (beta-1,4-GalT) V, which contributes to the biosynthesis of highly branched N-linked oligosaccharides characteristic of cancer cells. Our previous study showed that expression of the human beta-1,4-GalT V gene is regulated by Sp1 (Sato, T., and Furukawa, K. (2004) J. Biol. Chem. 279, 39574-39583), and a subsequent study showed that the gene expression is also activated by Ets-1, a product of the oncogene (Sato, T., and Furukawa, K. (2005) Glycoconj. J. 22, 365). Herein we report the mechanism of beta-1,4-GalT V gene activation by these transcription factors. The gene expression and promoter activity of beta-1,4-GalT V increased when the ets-1 cDNA was transfected into A549 cells, which contain a small amount of Ets-1, but decreased dramatically when the dominant-negative ets-1 cDNA was transfected into HepG2 cells, which contain a large amount of Ets-1. Luciferase assays using deletion constructs of the beta-1,4-GalT V gene promoter showed that promoter region -116 to +22 is critical for the transcriptional activation of the gene by Ets-1. Despite the presence of one Ets-1-binding site, which overlapped the Sp1-binding site, electrophoretic mobility shift assays showed that the region bound preferentially to Sp1 rather than to Ets-1. To solve this problem, we examined the transcriptional regulation of the human Sp1 gene by Ets-1 and found that the gene expression and promoter activity of Sp1 are regulated by Ets-1 in cancer cells. Functional analyses of two Ets-1-binding sites in the Sp1 gene promoter showed that only Ets-1-binding site -413 to -404 is involved in the activation of the gene by Ets-1. These results indicate that Ets-1 enhances expression of the beta-1,4-GalT V gene through activation of the Sp1 gene in cancer cells.

Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.

Cartilage destruction leads to severe joint diseases, such as osteoarthritis and spinal disorders with back pain, and cartilage regeneration is very inefficient. A major component of the cartilage extracellular matrix is the proteoglycan aggrecan that contains approximately 100 chondroitin sulfate (CS) chains, which impart water absorption and resistance to compression. Here, we demonstrate that chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) plays a critical role in CS biosynthesis in cartilage. By in situ hybridization and real time reverse transcription-PCR of developing cartilage, CSGalNAcT-1 exhibited the highest level of expression. Its expression in chondrogenic ATDC5 cells correlated well with that of aggrecan core protein. In heterozygote and homozygote aggrecan-null cartilage where aggrecan transcription is decreased, CSGalNAcT-1 transcription diminished accordingly. Overexpression of the enzyme in chondrocytic cells further enhanced CS biosynthesis but not that of the aggrecan core protein, indicating that the enzyme activity is not saturated in the cells and that aggrecan synthesized in the overexpressing cells is heavier than the native molecule. Analysis of the CS chains synthesized in the overexpressing cells by gel chromatography and that of disaccharide composition revealed that the CS chains had similar length and sulfation patterns. Furthermore, adenoviral gene delivery of the enzyme into intervertebral discs displayed a substantial increase in the level of CS biosynthesis. These observations indicate that CSGalNAcT-1 overexpression increases the number of CS chains attached to aggrecan core protein. Our studies may lead to a new therapeutic intervention, ameliorating the outcome of cartilage degenerative diseases.

Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.