Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme

Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34–72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR⁷²↓, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum.     

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu¹⁸³, Ser²⁴⁴, and Arg²⁸⁸ impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88_151–189 and GFP-MyD88_168–189), comprising the Glu¹⁸³ residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.     

Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau

Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I₂^PP2A). In AD brain, I₂^PP2A is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I₂^PP2A containing a conserved basic motif, ¹⁷⁹RKR¹⁸¹, which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I₂^PP2A in which the NLS was inactivated by ¹⁷⁹RKR¹⁸¹ → AAA along with ¹⁶⁸KR¹⁶⁹ → AA mutations. In this model, the mutant NLS (mNLS)-I₂^PP2A (I₂^PP2AAA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I₂^PP2A activated CAMKII and GSK-3β, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I₂^PP2A with PP2A and GSK-3β but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I₂^PP2A-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology.     

Dentin sialophosphoprotein (DSPP) is cleaved into its two natural dentin matrix products by three isoforms of bone morphogenetic protein-1 (BMP1)

The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPP^MQΔIE during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low-salt buffer (≤ 50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2's reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.     

Solution Structure of Proinsulin: CONNECTING DOMAIN FLEXIBILITY AND PROHORMONE PROCESSING*

The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic β-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (A- and B-domains); the tethered connecting (C) domain (as probed by {¹H}-¹⁵N nuclear Overhauser enhancements) is progressively less ordered. Although the BC junction is flexible, residues near the CA junction exhibit α-helical-like features. Relative to canonical α-helices, however, segmental ¹³C_α/β chemical shifts are attenuated, suggesting that this junction and contiguous A-chain residues are molten. We propose that flexibility at each C-domain junction facilitates prohormone processing. Studies of protease SPC3 (PC1/3) suggest that C-domain sequences contribute to cleavage site selection. The structure of proinsulin provides a foundation for studies of insulin biosynthesis and its impairment in monogenic forms of diabetes mellitus.     

Human Substance P Receptor Expressed in Sf9 Cells Couples with Multiple Endogenous G Proteins

Abstract

To identify the G proteins involved in the function of human substance P receptor (hSPR), the receptor was expressed in Sf9 cells using the baculovirus expression system. Maximal hSPR expression was up to 65 pmol/mg membrane protein. The following data indicated that hSPR in Sf9 membranes is coupled to endogenous G proteins: 1) binding of agonist radioligand [¹²⁵I]BHSP to the receptor was sensitive to guanine nucleotides; and 2) stimulation of the receptor increased [³⁵S]GTPγS binding. The hSPR-associated G proteins were identified by photoaffinity labeling with [α-³²P]-azidoanilido GTP ([α-³²P]AAGTP), followed by immunoprecipitation of the labeled G proteins with antibodies specific for various Gα-subunits. These experiments showed that stimulation of hSPR in Sf9 membranes activated multiple endogenous G proteins including Gα_o, Gα^q/11, and Gα. While hSPR's ability to associate with G^q/11 is well-documented, the present study provides the first evidence of hSPR's potential to activate Gα_o and Gα_s.     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2^pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2^pro activity by the NS3 protease domain. NS2^pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2^pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2^pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2^pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2^pro activity mimic structural changes occurring during NS3-mediated NS2^pro activation. Introducing these activating NS2^pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2^pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2^pro activation by NS3.     

Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.     

Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies

Background

Dentin sialophosphoprotein (DSPP) is the largest member of the SIBLING family and is the most abundant noncollagenous protein in dentin. DSPP is also expressed in non-mineralized tissues including metabolically active ductal epithelia and some cancers. Its function, however, is poorly defined. The carboxy-terminal fragment, dentin phosphoprotein (DPP) is encoded predominantly by a large repetitive domain that requires separate cloning/sequencing reactions and is, therefore, often incomplete in genomic databases. Comparison of DPP sequences from at least one member of each major branch in the mammalian evolutionary tree (including some "toothless" mammals) as well as one reptile and bird may help delineate its possible functions in both dentin and ductal epithelia.

Results

The BMP1-cleavage and translation-termination domains were sufficiently conserved to permit amplification/cloning/sequencing of most species' DPP. While the integrin-binding domain, RGD, was present in about half of species, only vestigial remnants of this tripeptide were identified in the others. The number of tandem repeats of the nominal SerSerAsp phosphorylation motif in toothed mammals (including baleen whale and platypus which lack teeth as adults), ranged from ~75 (elephant) to >230 (human). These repeats were not perfect, however, and patterns of intervening sequences highlight the rapidity of changes among even closely related species. Two toothless anteater species have evolved different sets of nonsense mutations shortly after their BMP1 motifs suggesting that while cleavage may be important for DSPP processing in other tissues, the DPP domain itself may be required only in dentin. The lizard DSPP had an intact BMP1 site, a remnant RGD motif, as well as a distinctly different Ser/Asp-rich domain compared to mammals.

Conclusions

The DPP domain of DSPP was found to change dramatically within mammals and was lost in two truly toothless animals. The defining aspect of DPP, the long repeating phosphorylation domain, apparently undergoes frequent slip replication and recombination events that rapidly change specific patterns but not its overall biochemical character in toothed animals. Species may have to co-evolve protein processing mechanisms, however, to handle increased lengths of DSP repeats. While the RGD domain is lost in many species, some evolutionary pressure to maintain integrin binding can be observed.     

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9

The induction of apoptosis by azadirachtin, a well‐known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC₅₀ at 48 and 72 h was 2.727 × 10⁻⁶ and 6.348 × 10⁻⁹ μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase‐1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase‐dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.     

Identification of the interaction sites of Inhibitor-3 for protein phosphatase-1

Inhibitor-3 is a potent inhibitor of protein phosphatase-1, with an IC₅₀ in the nanomolar range for the inhibition of the dephosphorylation of phosphorylase a. Human Inhibitor-3 possesses a putative protein phosphatase-1 binding motif, ³⁹KKVEW⁴³. We provide direct evidence that this sequence is involved in PP1 interaction by examining the effects of site-directed mutations of Inhibitor-3 on its ability to inhibit protein phosphatase-1. A second interaction site whose deletion led to loss of inhibitory potency was identified between residues 65 and 77. The existence of two interaction sites is consistent with the high inhibitory potency of Inhibitor-3, and with current models for other inhibitor and targeting proteins that interact with protein phosphatase-1 with high affinity.     

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.