Structure-Function Elucidation of a New α-Conotoxin,Lo1a,from Conus longurionis

α-Conotoxins are peptide toxins found in the venom of marine cone snails and potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors forming ligand-gated ion channels in the plasma membranes of certain neurons and the neuromuscular junction. Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson disease, and Alzheimer disease. To expand the knowledge concerning cone snail toxins, we examined the venom of Conus longurionis. We isolated an 18-amino acid peptide named α-conotoxin Lo1a, which is active on nAChRs. To the best of our knowledge, this is the first characterization of a conotoxin from this species. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs expressed in Xenopus laevis oocytes. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. Lo1a, a member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α₇ nAChRs with an IC₅₀ of 3.24 ± 0.7 μm. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. Because Lo1a has an unusual C terminus, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, to investigate the influence of the C-terminal residue. Lo1a-ΔD has a C-terminal Asp deletion, whereas in Lo1a-RRR, a triple-Arg tail replaces the Asp. They blocked the neuronal nAChR α₇ with a lower IC₅₀ value, but remarkably, both adopted affinity for the muscle subtype α₁β₁δϵ.     

Nogo receptor 3, a paralog of Nogo-66 receptor 1 （NgR1）, may function as a NgR1 co-receptor for Nogo-66

Nogo-A is a major myelin associated inhibitor that blocks regeneration of injured axons in the central nervous system (CNS).Nogo-66 (a 66-residue domain of Nogo-A) expressed on the surface of oligodendrocytes has been shown to directly interact with Nogo-66 receptor 1 (NgR1).A number of additional components of NgR1 receptor complex essential for its signaling have been uncovered.However,detailed composition of the complex and its signaling mechanisms remain to be fully elucidated.In this study,we show that Nogo receptor 3 (NgR3),a paralog of NgR1,is a binding protein for NgR1.The interaction is highly specific because other members of the reticulin family,to which Nogo-A belongs,do not bind to NgR3.Neither does NgR3 show any binding activity with Nogo receptor 2 (NgR2),another NgR1 paralog.Majority of NgR3 domains are required for its binding to NgR1.Moreover,a truncated NgR3 with the membrane anchoring domain deleted can function as a decoy receptor to reverse neurite outgrowth inhibition caused by Nogo-66 in culture.These in vitro results,together with previously reported overlapping expression profile between NgR1 and NgR3,suggest that NgR3 may be associated with NgR1 in vivo and that their binding interface may be targeted for treating neuronal injuries.     

RASgrf1, a Potential Methylatic Mediator of Anti-epileptogenesis?

Epileptogenesis, induced by status epilepticus (SE), is a chronic process, and intervention in this progress may prevent chronic epilepsy. It has been proposed that DNA methylation might be related with epileptogenesis. RASgrf1 has a differentially methylated region at the promoter which can silence gene expression. We have previously observed the down-regulation of RASgrf1 in epilepsy patients and proved that hypermethylation of RASgrf1 reaches maximal level at the latent period in mice after kainate-induced SE (KA mice), with corresponding alteration of RASgrf1 expression. In the present study, N-phthalyl-L-tryptophan (RG108), a DNA methyltransferase inhibitor, was applied in KA mice at latent phase and the behavior, electroencephalogram and pathological changes were observed in chronic phase. Methylation and expression of RASgrf1 were determined by polymerase chain reaction (PCR), western blotting, and bisulfite sequencing PCR. The results showed that the incidence of spontaneous recurrent seizures (SRS) was significantly lower in the RG108 group than the normal saline (NS) group. Subgroup analysis showed significant hypermethylation and lower expression of RASgrf1 in the RG108–SRS subgroup and the NS–SRS subgroup but not in the RG108–NSRS (no SRS) subgroup and the NS–NSRS subgroup compared with the control group. No significant difference was found between the RG108–SRS and NS–SRS subgroups. Meanwhile, hippocampal neuronal loss was observed in RG108–SRS and NS–SRS subgroups. We thus demonstrated that RG108 could modify the progression of epileptogenesis after KA induced SE and prevent chronic epilepsy. Meanwhile, hypermethylation of RASgrf1 after KA induced SE could be reversed with corresponding changes of RASgrf1 expression. Additionally, we speculated that RASgrf1 might be a potential epigenetic mediator in epileptogenesis and chronic epilepsy.     

The 3′ Overhangs at Tetrahymena thermophila Telomeres Are Packaged by Four Proteins,Pot1a,Tpt1, Pat1, and Pat2

Although studies with the ciliate Tetrahymena thermophila have played a central role in advancing our understanding of telomere biology and telomerase mechanisms and composition, the full complement of Tetrahymena telomere proteins has not yet been identified. Previously, we demonstrated that in Tetrahymena, the telomeric 3′ overhang is protected by a three-protein complex composed of Pot1a, Tpt1, and Pat1. Here we show that Tpt1 and Pat1 associate with a fourth protein, Pat2 (Pot1 associated Tetrahymena 2). Mass spectrometry of proteins copurifying with Pat1 or Tpt1 identified peptides from Pat2, Pot1a, Tpt1, and Pat1. The lack of other proteins copurifying with Pat1 or Tpt1 implies that the overhang is protected by a four-protein Pot1a-Tpt1-Pat1-Pat2 complex. We verified that Pat2 localizes to telomeres, but we were unable to detect direct binding to telomeric DNA. Cells depleted of Pat2 continue to divide, but the telomeres exhibit gradual shortening. The lack of growth arrest indicates that, in contrast to Pot1a and Tpt1, Pat2 is not required for the sequestration of the telomere from the DNA repair machinery. Instead, Pat2 is needed to regulate telomere length, most likely by acting in conjunction with Pat1 to allow telomerase access to the telomere.     

Recognition Mechanism of a Novel Gabapentinoid Drug,Mirogabalin, for Recombinant Human α2δ1, a Voltage-Gated Calcium Channel Subunit

Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α₂δ1. Here, to reveal the mirogabalin recognition mechanisms of α₂δ1, we present structures of recombinant human α₂δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α₂δ1 to those of the α₂δ2, α₂δ3, and α₂δ4 isoforms, of which α₂δ3 and α₂δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α₂δ1 ligand recognition.     

Rab21, a Novel PS1 Interactor,Regulates γ-Secretase Activity via PS1 Subcellular Distribution

γ-Secretase has been a therapeutical target for its key role in cleaving APP to generate β-amyloid (Aβ), the primary constituents of senile plaques and a hallmark of Alzheimer’s disease (AD) pathology. Recently, γ-secretase-associating proteins showed promising role in specifically modulating APP processing while sparing Notch signaling; however, the underlying mechanism is still unclear. A co-immunoprecipitation (Co-IP) coupled with mass spectrometry proteomic assay for Presenilin1 (PS1, the catalytic subunit of γ-secretase) was firstly conducted to find more γ-secretase-associating proteins. Gene ontology analysis of these results identified Rab21 as a potential PS1 interacting protein, and the interaction between them was validated by reciprocal Co-IP and immunofluorescence assay. Then, molecular and biochemical methods were used to investigate the effect of Rab21 on APP processing. Results showed that overexpression of Rab21 enhanced Aβ generation, while silencing of Rab21 reduced the accumulation of Aβ, which resulted due to change in γ-secretase activity rather than α- or β-secretase. Finally, we demonstrated that Rab21 had no effect on γ-secretase complex synthesis or metabolism but enhanced PS1 endocytosis and translocation to late endosome/lysosome. In conclusion, we identified a novel γ-secretase-associating protein Rab21 and illustrate that Rab21 promotes γ-secretase internalization and translocation to late endosome/lysosome. Moreover, silencing of Rab21 decreases the γ-secretase activity in APP processing thus production of Aβ. All these results open new gateways towards the understanding of γ-secretase-associating proteins in APP processing and make inhibition of Rab21 a promising strategy for AD therapy.     

Cleavage, a real turn-off? HIV-mediated proteolysis of PABP1

In this issue of the Biochemical Journal, Alvarez and colleagues have identified PABP1 [poly(A)-binding protein 1] as a target of protease cleavage during HIV infection. The study shows that HIV-1, HIV-2 and mouse mammary tumour virus, but not other retroviruses, target PABP1 for cleavage and identifies cleavage sites within the RNA-recognition motifs and C-terminal region of the protein. This suggests that PABP1 cleavage may be important in the shut-off of host translation during HIV infection. This extends the viral families that are known to target PABP1 to include Retroviridae, suggesting that PABP1 may be a central target of viral infection.     

Characterization of a cDNA encoding a protein with limited similarity to β1, 3-N-acetylglucosaminyltransferase

Glycosyltransferases constitute a large group of enzymes that are involved in a wide range of functions in all living organisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human putative glycosyltransferase gene named beta3GnTL1. Its cDNA is 1372 base pair in length, encoding a predicted protein with 361 amino acid residues. The human beta3GnTL1 is located to chromosome 17q25.3 by comparison of its cDNA with human gemome database. RT-PCR result shows the beta3GnTL1 is expressed at various levels in most of tissues examined.     

Plumieribetin, a Fish Lectin Homologous to Mannose-binding B-type Lectins, Inhibits the Collagen-binding ��1��1 Integrin

Recently, a few fish proteins have been described with a high homology to B-type lectins of monocotyledonous plants. Because of their mannose binding activity, they have been ascribed a role in innate immunity. By screening various fish venoms for their integrin inhibitory activity, we isolated a homologous protein from the fin stings and skin mucus of the scorpionfish (Scorpaena plumieri). This protein inhibits α1β1 integrin binding to basement membrane collagen IV. By protein chemical and spectroscopic means, we demonstrated that this fish protein, called plumieribetin, is a homotetramer and contains a high content of anti-parallel β strands, similar to the mannose-binding monocot B-lectins. It lacks both N-linked glycoconjugates and common O-glycan motifs. Despite its B-lectin-like structure, plumieribetin binds to α1β1 integrin irrespective of N-glycosylation, suggesting a direct protein-protein interaction. This interaction is independent of divalent cations. On the cellular level, plumieribetin failed to completely detach hepatocarcinoma HepG2 cells and primary arterial smooth muscle cells from the collagen IV fragment CB3. However, plumieribetin weakened the cell-collagen contacts, reduced cell spreading, and altered the actin cytoskeleton, after the compensating α2β1 integrin was blocked. The integrin inhibiting effect of plumieribetin adds a new function to the B-lectin family, which is known for pathogen defense.     

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit,Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca²⁺ release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490–524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490–508) is sufficient to reproduce activating effects of β1a490–524. Here we examined the effects of β1a490–508 on Ca²⁺ release and Ca²⁺ currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490–508 (25 nM) increased the peak Ca²⁺ release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490–508 in fibers. β1a490–508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490–508 peptide was used as negative control and produced negligible effects on Ca²⁺ release flux and RyR1 activity. Our results show that the β1a490–508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.     

TBX3, a downstream target of TGF-β1, inhibits mesangial cell apoptosis

Chronic kidney disease (CKD) is an increasingly common condition characterized by progressive loss of functional nephrons leading to renal failure. TGF-β1-induced mesangial cell (MC) phenotype alterations have been linked to the genesis of CKD. Here we show that TGF-β1 regulates TBX3 gene expression in MC. This gene encodes for two main isoforms, TBX3.1 and TBX3+2α. TBX3.1 has been implicated in cell immortalization, proliferation and apoptosis by inhibiting p14^ARF-Mdm2-p53 pathway, while TBX3+2α role has not been defined. We demonstrated that TBX3 overexpression abrogated MC apoptosis induced by serum deprivation. Moreover, we observed an enhancement in TBX3 protein expression both in glomerular and tubular regions in the model of 5/6 nephrectomy, temporally related to increased expression of TGF-β1, type IV collagen and fibronectin. Our results indicate that TBX3 acts as an anti-apoptotic factor in MC in vitro and may be involved in the mechanism by which TGF-β1 induces glomerulosclerosis and tubular fibrosis during the progression of nephropathies.     

Virulence as a consequence of genome instability of a novel temperate bacteriophage, ΦRsG1, of Rhodobacter sphaeroides Y

A novel temperate bacteriophage, designated RsG1, was isolated from Rhodobacter sphaeroides Y (previously designated Rhodopseudomonas sphaeroides) following exposure to mitomycin C. The phage morphology, as revealed from electron microscopy, showed a hexagonal head (90 by 46.5 nm) connected with a tail (116 by 9.4 nm), to which a collar was proximally attached. A morphologically similar phage was also produced by spontaneous lysis of the cells. While RsG1 did not grow on any other bacterial strain tested, spontaneously produced phage particles propagated (and formed plaques) on R. sphaeroides Y still carrying RsG1 in the prophage state. The genome of RsG1 consisted of double stranded linear DNA with cohesive ends and a GC-content of 71.8 mol%. The DNA molecules formed circles in vitro with a mean contour length of 17.18±0.4 m, which corresponds to a size of 49 kbase pairs (kb). On the other hand, DNA extracted from the virulent phage particles was heterogeneous and consisted of two DNA species of different size, occurring in a ratio of about 1:1. These molecules also circularized having contour lengths of 17.18±0.4 m and 14.02±0.41 m corresponding to 49 and 40 kb, respectively. Restriction digest analysis of the two DNA species and DNA from RsG1 indicated that they are similar, and allowed the indentification of an 11.5 kb EcoRI fragment that carries the cohesive ends. Because DNA from RsG1 and the 49 kb DNA of the virulent phage particles were indistinguishable with the criteria applied, it is suggested that phage particles containing the 40 kb DNA represent the virulent type of phage, termed RsG1.1.     

Tomato SlSnRK1 protein interacts with and phosphorylates βC1, a pathogenesis protein encoded by a geminivirus β-satellite

The βC1 protein of tomato yellow leaf curl China β-satellite functions as a pathogenicity determinant. To better understand the molecular basis of βC1 in pathogenicity, a yeast two-hybrid screen of a tomato (Solanum lycopersicum) cDNA library was carried out using βC1 as bait. βC1 interacted with a tomato SUCROSE-NONFERMENTING1-related kinase designated as SlSnRK1. Their interaction was confirmed using a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Plants overexpressing SnRK1 were delayed for symptom appearance and contained lower levels of viral and satellite DNA, while plants silenced for SnRK1 expression developed symptoms earlier and accumulated higher levels of viral DNA. In vitro kinase assays showed that βC1 is phosphorylated by SlSnRK1 mainly on serine at position 33 and threonine at position 78. Plants infected with βC1 mutants containing phosphorylation-mimic aspartate residues in place of serine-33 and/or threonine-78 displayed delayed and attenuated symptoms and accumulated lower levels of viral DNA, while plants infected with phosphorylation-negative alanine mutants contained higher levels of viral DNA. These results suggested that the SlSnRK1 protein attenuates geminivirus infection by interacting with and phosphorylating the βC1 protein.     

MUC1* Ligand,NM23-H1, Is a Novel Growth Factor That Maintains Human Stem Cells in a More Na?ve State

We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more “naïve” state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell''s inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell''s exit from pluripotency.