Arginine methylation next to the PY‐NLS modulates Transportin binding and nuclear import of FUS

Fused in sarcoma (FUS) is a nuclear protein that carries a proline‐tyrosine nuclear localization signal (PY‐NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY‐NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN‐binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN‐mediated nuclear import of ALS‐associated FUS mutants. The unmethylated arginine–glycine–glycine domain preceding the PY‐NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS‐FUS patients contain methylated FUS, while inclusions in FTLD‐FUS patients are not methylated. Together with recent findings that FUS co‐aggregates with two related proteins of the FET family and TRN in FTLD‐FUS but not in ALS‐FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.     

TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells

The RNA-binding proteins TDP-43 and FUS form abnormal cytoplasmic aggregates in affected tissues of patients with amyotrophic lateral sclerosis and frontotemporal lobar dementia. TDP-43 and FUS localize mainly in the nucleus where they regulate pre-mRNA splicing, but they are also involved in mRNA transport, stability, and translation. To better investigate their cytoplasmic activities, we applied an RNA immunoprecipitation and chip analysis to define the mRNAs associated to TDP-43 and FUS in the cytoplasmic ribonucleoprotein complexes from motoneuronal NSC-34 cells. We found that they bind different sets of mRNAs although converging on common cellular pathways. Bioinformatics analyses identified the (UG)(n) consensus motif in 80% of 3'-UTR sequences of TDP-43 targets, whereas for FUS the binding motif was less evident. By in vitro assays we validated binding to selected target 3'-UTRs, including Vegfa and Grn for TDP-43, and Vps54, Nvl, and Taf15 for FUS. We showed that TDP-43 has a destabilizing activity on Vegfa and Grn mRNAs and may ultimately affect progranulin protein content, whereas FUS does not affect mRNA stability/translation of its targets. We also demonstrated that three different point mutations in TDP-43 did not change the binding affinity for Vegfa and Grn mRNAs or their protein level. Our data indicate that TDP-43 and FUS recognize distinct sets of mRNAs and differently regulate their fate in the cytoplasm of motoneuron-like cells, therefore suggesting complementary roles in neuronal RNA metabolism and neurodegeneration.     

Enhancement of diphtheria toxin potency by replacement of the receptor binding domain with tetanus toxin C-fragment: a potential vector for delivering heterologous proteins to neurons

This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB(389)TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB(389)) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB(389)TTC was approximately 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB(389)MSH. The cytotoxic effect of DAB(389)TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB(389)TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB(389) catalytic fragment. These results suggest that a catalytically inactive form of DAB(389)TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons.     

Expression pattern and raft association of NIPSNAP3 and NIPSNAP4, highly homologous proteins encoded by genes in close proximity to the ATP-binding cassette transporter A1

The highly homologous genes NIPSNAP3 and NIPSNAP4, with 87% amino acid identity, are members of the NIPSNAP family with putative roles in vesicular trafficking. NIPSNAP3 mRNA and NIPSNAP4 mRNA and protein were detected in multiple tissues and cells at varying degrees. Interestingly, NIPSNAP3 is most highly expressed in skeletal muscle, where NIPSNAP4 has a low mRNA abundance. NIPSNAP4 was found associated with membranes and partly localized in rafts. The ubiquitous expression of the highly conserved NIPSNAPs and their association with membranes further support an important cellular function of these proteins probably linked to vesicular trafficking. The NIPSNAP3 and NIPSNAP4 genes are located in close proximity to the 3' end of the ATP-binding cassette transporter A1 (ABCA1), whose mutations cause familial high-density lipoprotein deficiency syndromes. The adjacent genomic location and the finding that ABCA1 is a regulator of vesicular trafficking may indicate a functional relation of these proteins, even though NIPSNAP4 does not interact directly with ABCA1 nor is its expression altered in cells with mutated ABCA1.     

Proposed mechanism for regulation of H2O2-induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H₂O₂-induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H₂O₂ treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.