Mutational analysis of Arabidopsis thaliana ABCE2 identifies important motifs for its RNA silencing suppressor function

• ATP‐binding cassette sub‐family E member 1 (ABCE1) is recognized as a strongly conserved ribosome recycling factor, indispensable for translation in archaea and eukaryotes, however, its role in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins (AtABCE1 and AtABCE2), sharing 81% identity. We previously reported that AtABCE2 functions as a suppressor of RNA silencing and that its gene is ubiquitously expressed. Here we describe the structural requirements of AtABCE2 for its suppressor function.
• Using agroinfiltration assays, we transiently overexpressed mutated versions of AtABCE2 together with GFP, to induce silencing in GFP transgenic Nicotiana benthamiana leaves. The influence of mutations was analysed at both local and systemic levels by in vivo imaging of GFP, Northern blot analysis of GFP siRNAs and observation of plants under UV light.
• Mutants of AtABCE2 with impaired ATP binding in either active site I or II failed to suppress GFP RNA silencing. Mutations disrupting ATP hydrolysis influenced the suppression of silencing differently at active site I or II. We also found that the N‐terminal iron–sulphur cluster domain of AtABCE2 is crucial for its suppressor function.
• Meaningfully, the observed structural requirements of AtABCE2 for RNA silencing suppression were found to be similar to those of archaeal ABCE1 needed for ribosome recycling. AtABCE2 might therefore suppress RNA silencing via supporting the competing RNA degradation mechanisms associated with ribosome recycling.

     

Macrophage migration inhibitory factor regulates joint capsule fibrosis by promoting TGF-β1 production in fibroblasts

Joint capsule fibrosis caused by excessive inflammation results in post-traumatic joint contracture (PTJC). Transforming growth factor (TGF)-β1 plays a key role in PTJC by regulating fibroblast functions, however, cytokine-induced TGF-β1 expression in specific cell types remains poorly characterized. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in inflammation- and fibrosis-associated pathophysiology. In this study, we investigated whether MIF can facilitate TGF-β1 production from fibroblasts and regulate joint capsule fibrosis following PTJC. Our data demonstrated that MIF and TGF-β1 significantly increased in fibroblasts of injured rat posterior joint capsules. Treatment the lesion sites with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP) reduced TGF-β1 production and relieved joint capsule inflammation and fibrosis. In vitro, MIF facilitated TGF-β1 expression in primary joint capsule fibroblasts by activating mitogen-activated protein kinase (MAPK) (P38, ERK) signaling through coupling with membrane surface receptor CD74, which in turn affected fibroblast functions and promoted MIF production. Our results reveal a novel function of trauma-induced MIF in the occurrence and development of joint capsule fibrosis. Further investigation of the underlying mechanism may provide potential therapeutic targets for PTJC.     

Unraveling the plant diversity of the Amazonian canga through DNA barcoding

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.     

Degradation of WTAP blocks antiviral responses by reducing the m6A levels of IRF3 and IFNAR1 mRNA

N ⁶‐methyladenosine (m⁶A) is a chemical modification present in multiple RNA species and is most abundant in mRNAs. Studies on m⁶A reveal its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. Although some recent discoveries indicate that m⁶A can affect the life cycles of numerous viruses as well as the cellular antiviral immune response, the roles of m⁶A modification in type I interferon (IFN‐I) signaling are still largely unknown. Here, we reveal that WT1‐associated protein (WTAP), one of the m⁶A “writers”, is degraded via the ubiquitination‐proteasome pathway upon activation of IFN‐I signaling. With the degradation of WTAP, the m⁶A levels of IFN‐regulatory factor 3 (IRF3) and interferon alpha/beta receptor subunit 1 (IFNAR1) mRNAs are reduced, leading to translational suppression of IRF3 and instability of IFNAR1 mRNA. Thus, the WTAP‐IRF3/IFNAR1 axis may serve as negative feedback pathway to fine‐tune the activation of IFN‐I signaling, which highlights the roles of m⁶A in the antiviral response by dictating the fate of mRNAs associated with IFN‐I signaling.     

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.     

Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM–TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717–726 and 732–746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3 ns. The positioning of the helix–linker–helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.     

Impact of genetic variability in the ABCG2 gene on ABCG2 expression,function, and interaction with AT1 receptor antagonist telmisartan

The ATP-binding cassette transporter ABCG2 plays a prominent role in cardiovascular and cancer pathophysiology, is involved in the pathogenesis of gout, and affects pharmacokinetics of numerous drugs. Telmisartan, a widely used AT1 receptor antagonist, inhibits the transport capacity of ABCG2 and may cause drug–drug interactions, especially in individuals carrying polymorphism that facilitate the telmisartan–ABCG2 interaction. Thus, the aim of this study was to identify ABCG2 polymorphisms and somatic mutations with relevance for the telmisartan–ABCG2 interaction. For this purpose, a cellular system for the conditional expression of ABCG2 was established. ABCG2 variants were generated via site-directed mutagenesis. Interaction of telmisartan with these ABCG2 variants was investigated in HEK293-Tet-On cells using the pheophorbide A efflux assay. Moreover, expression of ABCG2 variants was studied in these cells. Importantly, protein levels of the Q141K and F489L variant were significantly reduced, a phenomenon that was partly reversed by pharmacological proteasome inhibition. Moreover, basal pheophorbide A efflux capacity of S248P, F431L, and F489L variants was significantly impaired. Interestingly, inhibition of ABCG2-mediated pheophorbide A transport by telmisartan was almost abolished in cells expressing the R482G variant, whereas it was largely increased in cells expressing the F489L variant. We conclude that the arginine residue at position 482 of the ABCG2 molecule is of major importance for the interaction of telmisartan with this ABC transporter. Furthermore, individuals carrying the F489L polymorphism may be at increased risk of developing adverse drug reactions in multi-drug regimens involving ABCG2 substrates and telmisartan.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

Observations of rotation within the FoF1-ATP synthase: deciding between rotation of the Foc subunit ring and artifact

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.