Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78–121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98–114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98–114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.     

Pigment Epithelium-Derived Factor (PEDF) Peptide Eye Drops Reduce Inflammation,Cell Death and Vascular Leakage in Diabetic Retinopathy in Ins2Akita Mice

Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60–77 (P60) and neuroprotective PEDF78–121 (P78) derivatives were applied to Ins2^Akita mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1–4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 μg/mL for P60 and 0.9 μg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.     

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2^−/− mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA₂ enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2^−/− photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2^−/− and Pnpla2^+/− mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.     

Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A

NBCe1-A and AE1 both belong to the SLC4 HCO₃⁻ transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala⁸⁰⁰–Lys⁹⁶⁷. Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met⁸⁵⁸ accessible to both biotin maleimide and TAMRA and Thr⁹²⁶–Ala⁹²⁹ only to TAMRA labeling. The intracellular surface contains a highly exposed (Met⁸¹³–Gly⁸²⁸) region and a cryptic (Met⁸⁸⁷–Arg⁹⁰⁴) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp⁹⁶⁰. Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro⁸⁶⁸–Leu⁹⁶⁷ (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.     

Mesenchymal stem cells overexpressing PEDF decrease the angiogenesis of gliomas

The present study is an exploration of a novel strategy to target a therapeutic gene to brain tumour tissues. In the present study, we evaluated the feasibility of using hMSCs (human mesenchymal stem cells) to deliver PEDF (pigment epithelium-derived factor), a potent inhibitor of tumour angiogenesis, in a model of intracranial gliomas. To assess its potential of tracking gliomas, MSCs (mesenchymal stem cells) were injected into the cerebral hemisphere and it showed that MSCs infiltrated into the vessel beds and scattered throughout the tumour. In vitro migration assay showed that the VEGF (vascular endothelial growth factor) enhanced MSC migration. In contrast, the migratory activity of MSCs was significantly inhibited with the presence of PEDF. Systematic delivery of AAV (adeno-associated virus)–PEDF to established glioma xenografts resulted in increased apoptosis of gliomas. In addition, MSC–PEDF treatment prolonged the survival of mice bearing U87 gliomas. Taken together, these data validate that MSCs–PEDF can migrate and deliver PEDF to target glioma cells, which may be a novel and promising therapeutic approach for refractory brain tumour.     

The production and characterization of monoclonal antibodies against enkephalins

The hybridoma technology of Kohler and Milstein (1975) was utilized to produce monoclonal antibodies against the enkephalins. Two hybridomas, AD4 and DB4, produced monoclonal antibodies of the IgG type 1 class against Leu⁵-enkephalin that were highly specific for Leu⁵- and Met⁵-enkephalin. AD4 exhibited almost equal reactivity with either Leu⁵- or Met⁵-enkephalin, whereas DB4 exhibited only a 20% cross-reactivity with Met⁵-enkephalin. The IC₅₀ of these monoclonal antibodies were approximately two orders of magnitude greater than the IC₅₀ a polyclonal antiserum against enkephalins (A206; Miller et al 1978) used routinely in many immunochemical and immunocytochemical studies.The monoclonal antibodies, AD4 and DB4, exhibited specific sequence and size requirements for binding enkephalin-related peptides. The amino acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for recognition by AD4 and DB4. However, Tyr-Gly-Gly-Phe which lacks Leu or Met in the fifth position did not react with our monoclonal antibodies. Moreover, enkephalin-related peptides in which the enkephalin sequence was situated at the amino terminus and which contained six or more amino acids did not react significantly with AD4 or DB4. In particular, unlike the polyclonal antiserum A206, our monoclonal antibodies do not react with dynorphins 1–6 or 1–13. However, when the monoclonal antibody (AD4) was used to localize immunohistochemically the population of enkephalinergic amacrine cells in the chicken retina, it provided a staining pattern quite comparable to that observed in previous studies (Watt et al., 1983) using the polyclonal enkephalin antiserum A206. This finding therefore demonstrates that the immunoreactive products visualized in the enkephalin-immunoreactive amacrine cells of the chicken retina with the polyclonal antiserum correspond to authentic enkephalin or peptides very closely related to the enkephalins.     

In vitro cyclic AMP-mediated lipolytic activity of endorphins, enkephalins and naloxone

The maximum lipolytic activity (L_max) of β-endorphin is two and one half times that of Leu⁵-enkephalin and twice that of Met⁵-enkephalin, D-Ala², Met⁵-enkephalinamide, α-endorphin and γ-endorphin in the rabbit adipocyte. D-Met², Pro⁵-enkephalin-amide, however, has an L_max 1.6 times greater than that of Met⁵-enkephalin. The potencies (A₅₀) of Met⁵-enkephalin and its analogs and that of Leu⁵-enkephalin lie between 1.4 and 3 μM. The A₅₀ values for α-endorphin, β-endorphin and γ-endorphin are significantly less (1.2 × 10^?1 μM). Naloxone acts as an agonist in this system (A₅₀ = 2.5 μM; L_max 1.4 × Met⁵-enkephalin). All of the peptides and naloxone stimulated adenylate cyclase activity.     

Computational prediction and experimental characterization of a “size switch type repacking” during the evolution of dengue envelope protein domain III (ED3)

Dengue viruses (DEN) are classified into four serotypes (DEN1-DEN4) exhibiting high sequence and structural similarities, and infections by multiple serotypes can lead to the deadly dengue hemorrhagic fever. Here, we aim at characterizing the thermodynamic stability of DEN envelope protein domain III (ED3) during its evolution, and we report a structural analysis of DEN4wt ED3 combined with a systematic mutational analysis of residues 310 and 387. Molecular modeling based on our DEN3 and DEN4 ED3 structures indicated that the side-chains of residues 310/387, which are Val³¹⁰/Ile³⁸⁷ and Met³¹⁰/Leu³⁸⁷ in DEN3wt and DEN4wt, respectively, could be structurally compensated, and that a “size switch type repacking” might have occurred at these sites during the evolution of DEN into its four serotypes. This was experimentally confirmed by a 10 °C and 5 °C decrease in the thermal stability of, respectively, DEN3 ED3 variants with Met³¹⁰/Ile³⁸⁷ and Val³¹⁰/Leu³⁸⁷, whereas the variant with Met³¹⁰/Leu³⁸⁷, which contains a double mutation, had the same stability as the wild type DEN3. Namely, the Met310Val mutation should have preceded the Leu387Ile mutation in order to maintain the tight internal packing of ED3 and thus its thermodynamic stability. This view was confirmed by a phylogenetic reconstruction indicating that a common DEN ancestor would have Met³¹⁰/Leu³⁸⁷, and the intermediate node protein, Val³¹⁰/Leu³⁸⁷, which then mutated to the Val³¹⁰/Ile³⁸⁷ pair found in the present DEN3. The hypothesis was further confirmed by the observation that all of the present DEN viruses exhibit only stabilizing amino acid pairs at the 310/387 sites.     

Identification of a lipase-linked cell membrane receptor for pigment epithelium-derived factor

Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDF-binding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDF-binding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A(2) (PLA(2))zeta and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA(2)/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A(2) activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A(2) activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.     

Correlation between extracellular polysaccharide composition and nodulating ability inRhizobium trifolii

Summary Two auxotrophic mutants ofRhizobium trifolii which are deficient in nodulating ability have been isolated. Both mutants (strain RS 164 His^– and strain RS213 Leu^–) appear to synthesize abnormal extracellular polysaccharides as compared with the wild type strain RS 55. Simultaneous recovery of nodulating ability and wild type polysaccharide composition has been found in a Leu⁺ revertant of strain RS 213.Abbreviation EPS Extracellular Polysaccharide - NIG N-Methyl-N-Nitro-N-Nitrosoguanidine     

A+-Helix of Protein C Inhibitor (PCI) Is a Cell-penetrating Peptide That Mediates Cell Membrane Permeation of PCI

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His¹–Arg¹¹) and mPCI (Arg¹–Ala¹⁸) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI.     

Tn5-Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi

Escherichia coli (2492/pJB4JI) matings with Erwinia chrysanthemi produced kanamycin resistant (Km^r) transconjugants, a majority of which were gentamicin sensitive (Gm^s). A small proportion (about 0.8%) of the Km^r Gm^s clones were either auxotrophic or failed to catabolize galacturonate (Gtu⁻). The R plasmid (pJB4JI) DNA was detected in the parent E. coli strain and in a Km^r Gm^r transconjugant, but not in Km^r Gm^sE. chrysanthemi strains carrying Tn5-induced mutations. In Hfr crosses, Km^r (Tn5) was found linked with most mutations. A majority (>95%) of prototrophic recombinants were Km^s, except for Leu⁺ and Arg⁺ recombinants which were 30 to 50% Km^s. Spontaneous revertants were obtained for all markers except car, gtu, lys, thr, and trp. Prototrophic revertants, with the exception of Met⁺, Leu⁺, or His⁺ clones, were Km^s. We conclude from both genetic and physical data that Tn5 transposed from pJB4JI into different sites on the chromosome of E. chrysanthemi.     

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

CaBP4 modulates Ca²⁺-dependent activity of L-type voltage-gated Ca²⁺ channels (Cav1.4) in retinal photoreceptor cells. Mg²⁺ binds to the first and third EF-hands (EF1 and EF3), and Ca²⁺ binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg²⁺-bound and Ca²⁺-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1–99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg²⁺ or Ca²⁺ bound at EF1. The C-lobe binds Ca²⁺ at EF3 and EF4 and exhibits a Ca²⁺-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca²⁺-bound CaBP4 (Phe¹³⁷, Glu¹⁶⁸, Leu²⁰⁷, Phe²¹⁴, Met²⁵¹, Phe²⁶⁴, and Leu²⁶⁸) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca²⁺-dependent inactivation domain.     

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase,Acylpeptidyl Oligopeptidase,Releases N-Acylated Di- and Tripeptides from Oligopeptides

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser⁶¹⁵ and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9 family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The k_cat/K_m value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 μm⁻¹ s⁻¹, optimal pH was 7–8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met¹⁶–Glu¹⁰¹). Three-dimensional modeling revealed the three domain structures (residues Met¹⁶–Ala¹²⁶, which has no similar homologue with known structure; residues Leu¹²⁷–Met⁴⁹⁵ (β-propeller domain); and residues Ala⁴⁹⁶–Phe⁷³⁶ (α/β-hydrolase domain)) and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides.     

Identification of C-terminal Phosphorylation Sites of N-Formyl Peptide Receptor-1 (FPR1) in Human Blood Neutrophils

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 M_r) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu³¹²–Arg³²² and Arg³²³–Lys³⁵⁰) and extracellular FPR1 peptide (Ile¹⁹¹–Arg²⁰¹) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala³²³–Lys³⁵⁰ only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr³²⁵, Ser³²⁸, Thr³²⁹, Thr³³¹, Ser³³², Thr³³⁴, and Thr³³⁹. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC₅₀ = 33 ± 8 to 74 ± 21 nm. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.     

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNA^His possess an essential extra G_–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G_–1 is added by a tRNA^His guanylyl transferase. In prokaryotes, G_–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G_–1 we find here that both maturation pathways can be used. Indeed, tRNA^His with or without a G_–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA^His precursors at both positions G₊₁ and G_–1. The G_–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA^His without G_–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA^His with a G_–1 are recovered. This shows that a previously unreported tRNA^His guanylyltransferase activity is present in plant mitochondria.     

Binding of pigment epithelium-derived factor (PEDF) to retinoblastoma cells and cerebellar granule neurons. Evidence for a PEDF receptor.

Pigment epithelium-derived factor (PEDF) has neuronal differentiation and survival activity on retinoblastoma and cerebellar granule (CG) cells. Here, we investigated the presence of PEDF receptors on retinoblastoma Y-79 and CG cells. PEDF radiolabeled with (l25)I remained biologically active and was used for radioligand binding analysis. The binding was saturable and specific to a single class of receptors on both cells and with similar affinities (K(d) = 1.7-3.6 nM, B(max) = 0.5-2.7 x 10(5) sites/Y-79 cell; and K(d) = 3.2 nM, B(max) = 1.1 x 10(3) sites/CG cell). A polyclonal antiserum to PEDF, previously shown to block the PEDF neurotrophic activity, prevented the (125)I-PEDF binding. We designed two peptides from a region previously shown to confer the neurotrophic property to human PEDF, synthetic peptides 34-mer (positions 44-77) and 44-mer (positions 78-121). Only peptide 44-mer competed for the binding to Y-79 cell receptors (EC(50) = 5 nM) and exhibited neuronal differentiating activity. PEDF affinity column chromatography of membrane proteins from both cell types revealed a PEDF-binding protein of approximately 80 kDa. These results are the first demonstration of a PEDF-binding protein with characteristics of a PEDF receptor and suggest that the region comprising amino acid positions 78-121 of PEDF might be involved in ligand-receptor interactions.     

Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH₂-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K_i value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn³²–Met³⁸ and Gly⁹⁷–Leu¹⁰³, in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val³⁶-centerd hydrophobic cluster within the Asn³²–Met³⁸ region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile³⁵-Val³⁶-Tyr³⁷, in the Asn³²–Met³⁸ region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.     

Structural Studies of a Bacterial tRNAHIS Guanylyltransferase (Thg1)-Like Protein,with Nucleotide in the Activation and Nucleotidyl Transfer Sites

All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNA^His guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G₋₁ to the 5′-end of tRNA^His, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA^His. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA^His maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.     

Transmembrane Domain II of the Human Bile Acid Transporter SLC10A2 Coordinates Sodium Translocation

Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln⁷⁵, Phe⁷⁶, Met⁷⁹, Gly⁸³, Leu⁸⁶, Phe⁹⁰, and Asp⁹¹ in hASBT function. Computational analysis indicated that Asp⁹¹ may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr¹³⁴, Leu¹³⁸, and Thr¹⁴⁹) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln⁷⁵, Met⁷⁹, Thr⁸², and Leu⁸⁶ from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBT_NM), suggesting a universal mechanism for the SLC10A transporter family.