NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog

<p>Macroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete <i>Sordaria macrospora</i> (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of <i>Smnbr1</i> leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human <i>NBR1</i> homolog partially rescues the phenotypic defects of the fungal <i>Smnbr1</i> deletion mutant. The <i>Smnbr1</i> mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the <i>Smnbr1</i> deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.</p

C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease

<div><p>Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene <i>YHB1</i> resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of <i>YHB1</i>, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by αSyn di-tyrosine dimers. Our findings uncover a complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of αSyn that likely participates in PD pathology.</p></div

Determination of crosslinked peptides from αSyn and A30P.

<p>(A) Analysis of di-tyrosine dimers. Exemplary heat map diagram of the number (N) of identified di-tyrosine crosslinked peptides of the non-treated αSyn samples. (B) Distribution of all identified di-tyrosine peptides for αSyn. Identified combinations of crosslinked peptides are presented as percentage of n (n = total number of MS2 spectra verified as crosslinked peptides). (C) Distribution of all identified di-tyrosine peptides for A30P.</p

Tyrosine mutation of A30P decreases toxicity in <i>Δyhb1</i>.

<p>(A) Spotting analysis of αSyn, A30P, 4(Y/F) αSyn, A30P/4(Y/F) and GFP (control) expressed in <i>YHB1</i> and Δ<i>yhb1</i> yeast on non-inducing and galactose-inducing SC-Ura plates after 3 days of growth. (B) Cell growth analysis of <i>YHB1</i> and Δ<i>yhb1</i> yeast expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) and GFP (control) at time point 20 h. Significance of differences was calculated with t-test (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01, n = 3). (C) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing SC-Ura medium. Significance of differences was calculated with t-test (*, <i>p</i> < 0.05, **, <i>p</i> < 0.01, n = 6).</p

<i>dipA</i> gene locus and multiple alignment of its deduced metallophosphatase domain.

<p><b>(A)</b> Schematic view of the <i>dipA</i> (AN10946) gene locus, transcript (mRNA) and deduced DipA protein. White boxes correspond to three introns (I, II, III). The <u>m</u>etallo<u>p</u>hos<u>p</u>hatase domain (MPP) is highlighted in black and the domain of unknown function (DUF) in grey. <b>(B)</b> Multiple alignment of MPP consensus sequence (cl13995) with DipA from <i>Aspergillus nidulans</i> and related proteins from other organisms including <i>Aspergillus niger</i>, <i>Aspergillus oryzae</i>, <i>Penicillium roqueforti</i>, <i>Neurospora crassa</i>, <i>Ustilago maydis</i> and <i>Schizosaccharomyces pombe</i>. Asterisks: putative active site D51, H73 and D76 residues. Red: high (90%), blue: low (50%) consensus values [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005949#pgen.1005949.ref056" target="_blank">56</a>].</p

DenA-DipA cytoplasmatic movements <i>in vivo</i>.

<p><b>(A)</b> Fluorescence microscopy of DenA-GFP subpopulations within vegetative hyphae located the protein in the nucleus (N), in the cytoplasm and there with a specific enrichment at septa (S). Control: wild type without GFP. <b>(B)</b> Bimolecular fluorescence studies (BiFC) of DenA (<i>denA</i>::<i>nyfp</i>) and DipA (<i>dipA</i>::<i>cyfp</i>) showed restricted interaction in the cytoplasm, at septa (S) and close to, but not inside nuclei (N). The septal and the nuclear regions are enlarged (white squares; scale bar: 1 μm). Control: strain co-expressing <i>denA</i>::<i>nyfp</i> and <i>cyfp</i>, respectively. <b>(C)</b> Dynamic co-transport of DenA-DipA between nuclei and septa in time lapse of bimolecular fluorescence strain <i>denA</i>::<i>nyfp</i>-<i>dipA</i>::<i>cyfp</i> over 170 seconds. White arrows mark a single interaction complex. <b>(D</b>) Time lapse microscopy over 110 seconds of <i>denA</i>::<i>nyfp</i>-<i>dipA</i>::<i>cyfp</i> with stained mitochondria (red) with a white arrow marking single DenA-DipA. Expressed <i>rfp</i>::<i>h2A</i> decorates nuclei, membranes were stained with FM4-64 and mitochondria with MitoTracker. Scale bar: 5 μm.</p

The human <i>NGB</i> gene for neuroglobin alters A30P and αSyn aggregation in yeast and mammalian cells.

<p>(A) Spotting analysis of <i>YHB1</i> and Δ<i>yhb1</i> yeast cells co-expressing αSyn and GFP (control) with either empty vector as control or <i>YHB1</i> and <i>NGB</i>, respectively, on non-inducing and galactose-inducing SC-Ura medium after 3 days. (B) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing medium (n = 3). (C) Spotting analysis of <i>YHB1</i> and Δ<i>yhb1</i> yeast cells co-expressing A30P and GFP (control) with either empty vector (pME2788) as control or <i>YHB1</i> and <i>NGB</i>, respectively, on non-inducing and galactose-inducing SC-Ura medium after 3 days. (D) Quantification of the percentage of cells displaying A30P aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (**, <i>p</i> < 0.01, n = 3). (E) Fluorescence microscopy of H4 cells co-expressing SynT, synphilin-1 and pcDNA (control) or NGB-mCherry. Nuclei are stained with Hoechst dye (blue). Scale bar = 30 μm. (F) Quantification of the percentage of H4 cells displaying αSyn inclusions after 48 h after transfection. Cells were classified into three groups according to the number of αSyn-immunoreactive inclusions observed: cells with 10 inclusions, cells with less than 10 inclusions and cells without inclusions. Significance of differences was calculated with t-test (*, <i>p</i> < 0.05, n = 3). (G) Lactate dehydrogenase (LDH) activity measurements support that <i>NGB</i> is non-toxic for H4 cells. H4 cells transfected with empty mammalian expression vector pcDNA3.1, with empty pcDNA3.1 or pcDNA3.1 encoding neuroglobin-mCherry (<i>NGB</i>) together with SynT and synphilin-1 (SynT+Synphilin-1) were analyzed. Media from indicated H4 cells were collected and the secretion of lactate LDH was determined as a measure of cytotoxicity. Significance of differences was calculated with t-test (not significant (n.s.); n = 3).</p

<i>A</i>. <i>nidulans</i> strains constructed and used in this study.

<p><i>A</i>. <i>nidulans</i> strains constructed and used in this study.</p

Protein amount of DenA-GFP and amino acid substituted variants during development.

<p>Western hybridization with equal amounts of protein extracts of DenA-GFP (54.5 kDa) compared to <b>(A)</b> DenA^S253D-GFP carrying a negative charge reminiscent of a phosphorylated protein, <b>(B)</b> DenA^S253A-GFP which cannot be phosphorylated, <b>(C)</b> DenA^{S243D-S245D-S253D}-GFP with three negative charges mimicking a triple phosphorylated protein and <b>(D)</b> the corresponding DenA^{S243A-S245A-S253A}-GFP which cannot be phosphorylated. Samples were taken from vegetative hyphae (Veg) and at indicated time points (in hours) of illumination which induces asexual development (Asex). Membranes were treated with GFP-antibody to visualize the fusion protein and free GFP (25 kDa). Loading control: Ponceau staining. Lower panels show quantification of band intensities of DenA-GFP and its respective variants relative to vegetative growth.</p

Phenotypical characterization of mutant strains lacking functional DipA.

<p><b>(A)</b> Top and bottom view of point-inoculated wild type (WT), <i>dipA</i>* (codon exchange of catalytic core), <i>dipA</i> deletion (Δ<i>dipA</i>) and complementation (Compl.) strains incubated for three days under asexually development inducing conditions. Zoomed view represents binocular images of respective strains with asexual structures (conidiophores, co) and the sexual fruiting bodies (cleistothecia, cl) after seven days. Scale bar: 100 μm. <b>(B)</b> Colony diameter of point-inoculated asexually grown colonies measured for six days. The mean values with standard deviations derived from three independent experiments are shown. <b>(C)</b> Quantification of conidiospores after four days. The mean values with standard deviations from three independent experiments are shown. <b>(D)</b> Diagram illustrates distances between septa. Data derived from analyzing 70 hyphae of each strain. Shown are the mean values with standard deviations. <b>(E)</b> Fluorescence microscopy of hyphae of WT and <i>dipA</i> deletion strain. Membranes/septa were stained with FM4-64. White arrows are highlighting septa. Scale bar: 5 μm.</p