EMBO/soda-nel huggingface dataset:
Pyruvate kinase variant tunes carbon metabolism, growth and stress tolerance in fission yeast (2020)
Figure 1

Figure. 1: A variant in conserved pyk1 region is associated with increased sensitivity to antimycin A.

(A) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects (Materials and Methods). After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12.

(B) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70).

(C) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually.

(D) Sequence alignment of the region of interest in eukaryotic PYK proteins. The threonine residue at the highlighted position 343 is unique for the reference (laboratory) S. pombe strains. For this analysis, we collected 25 Pyk1 homologues from a wide variety of eukaryotes, including animals, plants and fungi, using the HomoloGene resource (Accession 37650). We manually expanded the homologous group to include three other species of the Schizosaccharomyces genus (Rhind et al, 2011). Sequences were aligned with MAFFT (Katoh et al, 2017) and visualised with Jalview (Waterhouse et al, 2009).

(E) Phylogenetic tree based on 31 biallelic SNPs in pyk1 and in 500bp up- and down-stream regions. Strains carrying the common A-allele are in orange, while strains carrying the unusual T-allele are in blue (strain names as in ref. (Jeffares et al, 2015)). Strains JB22 and JB50 (underlined and bold) refer to heterothallic and homothallic versions of the laboratory strain, respectively. The pie chart shows the relative allele frequencies at the genomic locus across all wild strains. Genotype calls at the locus were checked manually and were conclusive, i.e. no strain produced reads with both alleles.
Pyruvate kinase variant tunes carbon metabolism, growth and stress tolerance in fission yeast (2020)
Figure 2

Figure 2: Multi-omic functional investigation of Pyk1 variants.

(A) PCA of metabolite data based on concentrations of 27 central-carbon metabolism intermediates. To visualise this high-dimensional data set, we divided the concentration of each metabolite by the median concentration of the T-strain. This normalisation corrects for the large differences in concentrations observed between metabolites but maintains the relative variance within each metabolite. Biological replicates of the T- and A-strains show distinct profiles, largely driven by concentrations of phosphoenolpyruvate, 2-/3-phosphoglyceric acid, NADH and NADPH, as indicated by top loading vectors for each principal component. The biological repeats for the three edited A-strains (circles, squares and triangles) behave similarly, with a variance comparable to that of the biological replicates of the single T-strain.

(B) Left boxplot: two glycolytic intermediates directly upstream of PYK were strongly depleted in the A-strain (nT-strain=9, nA-strain=8). Right barplot: PYK activity was directly measured using a lactate dehydrogenase-coupled colorimetric enzyme activity assay, showing the mean and standard deviation of substrate conversion rate for 3 biological replicates, each measured in technical duplicates.

(C) Concentrations of fructose-1,6-bisphosphate, which correlate with glycolytic flux, are significantly higher in A-strain (ratio = 1.35, nT-strain=9, nA-strain=8).

(D) Boxplots for selected metabolomics data indicate differences in energy and redox status between T- and A-strains (nT-strain=9, nA-strain=8).

(E) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T- and A-strains (FDR <10%) and are measured in all four conditions (columns). First column: genes ordered by increasing fold-changes for RNAs (computed as log2[T]-log2[A]). Second column: fold-changes for proteins (computed as log2[T]-log2[A]). Third column: fold-changes for RNAs in cells treated with rapamycin & caffeine (TORC1 inhibition; computed as log2[treatment]-log2[control]) (data from ref. (Rallis et al, 2013)). Fourth column: fold-changes for RNAs in cells treated with H2O2 (oxidative stress; computed as log2[treatment]-log2[control]) (data from Chen et al, 2003). Log2 fold-changes are capped at absolute values of 1 for all columns.

(F) Gene Ontology (GO) enrichment analysis of differentially expressed transcripts (using all measured transcripts as background). Shown are Biological Process terms only, with additional plots for Molecular Function and Cellular Component terms in Appendix Fig. S5. The length of the bar represents the significance of the enrichment, while its color reflects the direction and magnitude of differential expression (A- vs T-strain) of individual genes annotated to this term.

Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)
A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i (2016)
Figure 1

Figure 1. Chemical structures of α’, β’ epoxyketone immunoproteasome inhibitors analyzed in this study.

The syntheses of the ligands PR-924, 9, 14, 16, 17 and 18 have been described previously (de Bruin et al, 2014). All ligands share the peptide scaffold shown in the upper left corner with P1, P2 and P3 side chains and an N-cap; they are equipped with a l-Ala (stereochemistry (S)) or d-Ala (stereochemistry (R)) residue at P3 (red) and either an N-acylmorpholine or a 3-methyl-1H-indene group (green). Furthermore, compounds differ in their P2 residue (either methoxytyrosine or tryptophane; blue). Inhibitor 9, also termed LU-015i, is endowed with an exceptional cyclohexyl residue (gray), which is associated with increased selectivity for human β5i.
A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i (2016)
Figure 2

Figure 2. Inhibition of the chymotrypsin-like activities of yeast, human and mouse proteasomes by ONX 0914, PR-924, 9, 14, 16, 17 and 18.

A. Inhibition of human β5c and β5i by ONX 0914 and PR-924. IC50 values are deduced from the fitted data.

B. IC50 values [µM] for all compounds towards the yeast, human and mouse β5 subunits.

For inhibition curves see Appendix Figure S2. The ratio IC50(β5c)/IC50(β5i), a measure of β5i selectivity, is given. Large values indicate high selectivity for β5i.

Data information: Genuine WT proteasomes were used for all inhibition assays. IC50 values [µM] were determined in triplicates with the fluorogenic substrate Suc-LLVY-AMC using an enzyme concentration of 13 nM. Mean standard deviations are indicated as bars and given in Appendix Table S2.
A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i (2016)
Figure 3

Figure 3. Expression of mammalian β5 proteasome subunits in yeast.

A. Schematic representation of yeast and mammalian β5 subunits and their propeptides. Secondary structure elements, helices (H) and sheets (S), are numbered. The full length human hβ5i (yellow) and β5c (red) as well as the mouse mβ5i (green) subunits cannot substitute the endogenous yeast yβ5 subunit (gray). Expression of the mammalian subunits with their natural propeptide (pp; colored) or with the yβ5 propeptide (pp; gray) is lethal in a yeast pre2Δ knock-out strain (for details see Appendix Supplementary Methods).

B. Chimeric β5 subunits are schematically illustrated according to panel A. The N-terminal part of the subunits, which forms the primed and non-primed substrate binding pockets and the active site surroundings, was taken from human β5i and the C-terminus from yeast. A construct featuring residues 1-173 from human β5i was still lethal. Replacement of the residues 1-139 of yβ5 by the human β5i counterpart (aa 1-138; see Appendix Supplementary Note) however was compatible with yeast survival. Based on this observation, analogous fragments of the human β5c and mouse β5i subunits were incorporated into the yβ5 subunit to create the respective hβ5c and mβ5i chimeras.

C. Subunit β6 significantly contributes to the S3 and S4 pockets of the ChT-L active site. In order to mimic the entire mammalian β5/6 substrate binding channel in yeast we mutated five residues of the yβ6 subunit as well (G89S, K90R, H98Y, T99N and E120Q). These mutations correspond to both the human and the mouse β6 sequence and create two mammalian segments aa 87-101 and aa 108-121 (blue; see also Appendix Supplementary Note).

D. The mouse specific point mutation V31M was inserted into the hβ5i/hβ6 chimera to investigate its impact on inhibitor binding.

E. Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 °C or 37 °C. Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity.

F. Surface illustration of the yCP. The β5 and β6 subunits are depicted in dark gray, with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue, respectively. The β5’ and β6’ subunits of the second β-ring are hidden on the back of 20S proteasome.
A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i (2016)
Figure 4

Figure 4. Crystal structures of chimeric human proteasomes in their apo state.

A. Superposition of the β5i/β6 active sites of genuine mouse iCP and the hβ5i/hβ6 human/yeast chimera illustrates that Met45 adopts the same ‘open’ conformation in both structures. Amino acid side chains important for substrate binding are depicted as sticks. The catalytic Thr1 is colored in black. Met31, the single amino acid difference between mouse and human β5i active sites, is highlighted in green.

B. Superposition of the genuine mouse and the human chimeric β5c/β6 active sites according to panel A proves the proper orientation of Met45 in the ‘closed’ conformation.

C. Superposition of the β5c/β6 and β5i/β6 active sites of the natural mouse cCP and iCP according to panel A. Note the distinct conformations of Met45 (Huber et al, 2012).

D. Superposition of the hβ5c/hβ6 and the hβ5i/hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C.

Data information: This figure contains previously published coordinates (Huber et al, 2012): mouse cCP (PDB accession code: 3UNE) and mouse iCP (PDB accession code: 3UNH).
A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i (2016)
Figure 5

Figure 5. Ligand complex structures of human chimeric proteasomes reveal distinct binding modes for β5c and β5i.

A. Complex structures of the human β5c/β6 (left) and β5i/β6 (right) chimeric proteasomes with PR-924. The ligand binds in a linear (l) manner to the β5c/β6 substrate binding channel, but in a kinked (k) conformation to the β5i/β6 active site. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ. The inhibitor and Thr1 have been omitted for phasing.

B. Comparison of the conformations of ONX 0914, PR-924, 14, 16, 17 and 18 bound to the chimeric hβ5/hβ6 substrate binding channels. The l amino acid compounds ONX 0914 and 14 display a linear (l) binding mode to the hβ5c/hβ6 (left) and the hβ5i/hβ6 (right) chimeras, whereas the ligands 16 and 17 (P3-d-Ala and morpholine cap) adopt a kinked (k) conformation in both β5 substrate binding channels. Remarkably, the orientation of PR-924 and its analogue 18 (P3-d-Ala and 3-methyl-1H-indene cap) in the hβ5c/hβ6 chimera (and in WT yβ5; see Appendix Figure S5)) differs from that observed with the hβ5i/hβ6 chimera. Note that the inhibitors are rotated by about 45 ° compared to panel A.
A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i (2016)
Figure 6

Figure 6. The impact of Met31 on ligand binding to the β5i active site.

A. Complex structures of the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right) with PR-924 visualize the kinked (k) orientation. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A. Steric hindrance of Met31 (green) with the bulky 3-methyl-1H-indene cap is indicated by black double arrows.

B. Superposition of inhibitors bound to the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right). Compounds display the same binding modes (kinked (k) or linear (l)) to the mβ5i/mβ6 active site as to the corresponding human β5i/β6 subunit (Fig. 5) and to the hβ5i-V31M/hβ6 mutant. Note that the inhibitors are rotated by about 45° compared to panel A.

C. Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3-d-Ala compounds PR-924 and 18. The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45. Residue 31 impacts on the position of the 3-methyl-1H-indene cap of PR-924 and 18. Due to steric hindrance, the N-cap is shifted in the mouse and in the hβ5i-V31M/hβ6 chimera compared to the hβ5i/hβ6 mutant proteasome crystal structures (black double arrow). Note that the structures are rotated by 90° compared to panel B.
A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i (2016)
Figure 7

Figure 7. Overview of key inhibitors and their distinct binding modes to the subunits yβ5, hβ5c, mβ5i and hβ5i.

ONX 0914 binds in a linear mode to all active sites. Changing the stereochemistry at the P3 site from l-Ala (S) to d-Ala (R) leads to a kinked conformation in the β5 substrate binding channels (compound 16). Substitution of the morpholine cap of 16 by a larger 3-metyhl-1H-indene moiety yields PR-924 and results in different inhibitor conformations at the yβ5/hβ5c and the mammalian β5i active sites. Hereby, the selectivity for hβ5i over hβ5c is drastically increased (IC50 ratio hβ5c/hβ5i: 255) compared to ONX 0914 (IC50 ratio hβ5c/hβ5i: 20) and compound 16 (IC50 ratio hβ5c/hβ5i: 5.2, Fig 2). Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR-924, thereby creating 7-fold selectivity for human β5i.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 1.

siRNA screen for PX domain proteins in autophagy. (A) HEK GFP-LC3 cells were transfected with siRNA pools targeting PX domain proteins and starved (large circles) or not starved (small circles) for 2 h, followed by fixation and counterstaining of the nuclei by Hoechst. The images were processed for high-content image analysis to quantify the number of GFP-LC3 spots per cell. The graph shows the average of three independent experiments in triplicate with a total of 30,000 cells. siRNA against ULK1 and TSG101 were used as controls (blue circles). Green, negative controls; red, PX domain proteins. (B) Selected PX domain proteins were depleted by transfection of HEK GFP-LC3 with the four individual siRNA oligos of the pools used in A. The cells were treated, imaged, and analyzed as in A. The area of each circle corresponds to the relative amount of target mRNA as measured by qPCR. The graph shows the average of two independent experiments in triplicate with a total number of 18,000 cells. Coloring is as in A. (C) Micrographs of starved HEK GFP-LC3 cells treated with nontargeting or siSNX18-3 siRNA. (left and middle) 3 × 3 fields of cells. (right) Close-up of a few cells from the respective population. The images in the right panels are shown again in Fig. S1 B alongside cells treated with other SNX18-targeting siRNAs. Bars, 10 µm. See also Table S1 and Table S2.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 2.

SNX18 is a positive regulator of autophagy. (A) HEK GFP-LC3 cells transfected with control (siCtrl) or two different SNX18 siRNA oligos (siSNX18-3 and siSNX18-5) were starved or not starved for 2 h in the presence or absence of BafA1. GFP-LC3 lipidation and SNX18 protein knockdown were monitored by immunoblotting. The graph shows the average GFP-LC3-II relative to actin normalized to siCtrl-1 fed ± SEM (error bars), n = 4. (B) The degradation of long-lived proteins in HeLa cells transfected with control or SNX18 siRNA was quantified after 4 h of starvation in the absence or presence of 3-MA and normalized to the degradation in fed cells (mean ± SEM [error bars], n = 3). (C) HEK GFP-LC3 cells were transfected with control or SNX18 siRNA and then with a myc control or siRNA-resistant myc-SNX18 plasmid. The number of GFP-LC3 spots per cell was quantified (graph shows mean ± SEM [error bars], n = 3). (D) HEK GFP-LC3 cells were transfected with myc-SNX18 or a myc control plasmid and treated as in A. The graph shows the average LC3-II levels relative to actin normalized to myc-transfected fed cells, ±SEM (error bars). n = 3. (E) HEK GFP-LC3 cells were transfected with control or Atg7 siRNA and then with myc-SNX18 or a myc control plasmid, followed by quantification of the number of GFP-LC3 spots per cell (graphs show mean ± SEM [error bars], n = 3). Representative images are shown. Bars, 5 µm. (F) The degradation of long-lived protein in HeLa cells transfected with myc-SNX18 or a myc control plasmid was quantified as in B (graph shows mean ± SEM [error bars], n = 4). *, P < 0.05; **, P < 0.01. See also Figs. S1 and S2.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 3.

SNX18 colocalizes with autophagic markers in the perinuclear area. (A) The total membrane fractions from fed or starved HeLa cells were analyzed by density gradient centrifugation and probed for the indicated proteins by immunoblotting. Migration of different organelles is indicated, based on the positions in the gradient of calnexin (ER), GM130 (Golgi stack/CGN), SNX9 (plasma membrane), AP-1, TfR and Rab11a (Endosomes/TGN), and LC3-II and GABARAP-II (autophagosomes). (B) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130. Panels to the right show enlarged views of the boxed regions. (C) HeLa cells were transfected with GFP-Atg14L and immunostained for SNX18 and calnexin. The boxed regions are enlarged on the top and bottom. (D and E) HEK GFP-DFCP1 cells were starved or not starved for 50 min and immunostained for SNX18 and LC3 (D) or WIPI2 (E). Results shown are mean ± SEM (error bars), n = 3 with 30 cells in each experiment. *, P < 0.05. (F) HeLa cells were transfected with control or SNX18 siRNA, starved for 2 h, and immunostained against SNX18 and Atg16L1. (G) HEK GFP-DFCP1 cells were transfected with control or SNX18 siRNA, starved for 50 min, and immunostained for Atg16L1. All images are from confocal microscopy. Inset panels show enlarged views of the boxed regions. Bars, 5 µm. See also Fig. S3.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 4.

SNX18 binding to PI(4,5)P2 membranes is required for its function in autophagy. (A) HeLa GFP-LC3 cells were transfected to express myc-SNX18, immunostained against myc, and analyzed by confocal imaging. Asterisks indicate a nontransfected cell. Bars: (main panels) 20 µm; (inset) 10 µm. (B) HeLa cells transfected with flag-Atg16L1 and myc-SNX18 were starved or not starved for 2 h before immunostaining with anti-myc and anti-flag antibodies and confocal microscopy. Bars, 5 µm. (C) SNX18 domain structure. An N-terminal SH3 domain is followed by a low-complexity region (LC). The membrane-remodeling part consists of an amphipathic helix (helix 0), a PX domain that is flanked by sequences forming a Yoke domain (Y), and a BAR half-domain. The functional BAR domain is formed by dimerization. Truncated constructs and point mutations used are indicated. (D) HeLa cells were transfected with the indicated myc-SNX18 plasmids for 6 h and starved for an additional 2 h before harvest. Equal parts of cytosol (C) and membrane (M) fractions were analyzed by Western blotting. The graph shows the average membrane-bound myc-SNX18 ± SEM (error bars), n = 3. (E and F) HEK GFP-LC3 cells were transfected with the indicated mCherry-SNX18 (E) or mycSNX18 (F) constructs for 16 h. The number of GFP-LC3 spots per transfected cell (E; graph shows mean ± SEM [error bars], n = 5) or ratio of LC3-II to actin determined by Western blotting (F) was quantified. (G and H) HEK GFP-LC3 cells transfected with myc-SNX18 or a myc control plasmid alone (G) or together with CFP-FRB and mRFP-FKBP or mRFP-FKBP 5pase (H) for 6 h were treated or not treated with 5 µM ionomycin (G) or 2.5 µM A/C heterodimerizer (rapalog; H) for the last 3 h before analysis of the total intensity of GFP-LC3 spots per transfected cell (graph shows mean ± SEM [error bars], n = 3). (I) MEFs were transfected with myc, myc-SNX18 WT, or KR for 16 h, starved or not starved for 2 h, and immunostained for endogenous Atg16L1. Insets show enlarged views of the boxed regions. Bars, 5 µm. (J) HeLa cells were transfected for 12 h with the indicated constructs and starved for 90 min. Cells were solubilized and immunoprecipitated with anti-flag or an unrelated antibody of the same isotype (mock ab). Immunoprecipitates and 2% of the lysates were analyzed by immunoblotting against myc and flag. *, P < 0.05; **, P < 0.01. See also Fig. S3.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 5.

The tubulation activity of SNX18 is important for its role in autophagy and is regulated by phosphorylation. (A) HeLa GFP-SNX18 cells were starved for the indicated time, followed by incubation in full medium (Refeed) where indicated. Cell lysates were analyzed for LC3 by immunoblotting, or immunoprecipitated with GFP-Trap before immunoblotting against SNX18(pS233) and GFP. The ratio of pS233 to GFP, and the ratio of LC3-II to LC3-I, was quantified, and normalized to 1 for the zero-time sample in both cases (graph shows mean ± SEM [error bars], n = 4; *, P < 0.05). (B) The indicated myc-SNX18 PX-BAR constructs were transfected into HeLa cells, and their tubulation efficiency was measured by scoring the percentage of cells displaying more than three elongated structures longer than 5 µm (n = 300). The graph shows the mean of two independent experiments ± range (error bars). Bars, 10 µm. (C) The indicated myc-tagged SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 5. (D) HeLa GFP-LC3 cells were transfected with the indicated myc-tagged SNX18 constructs for 16 h, starved for 2 h, and analyzed by confocal imaging. Bars, 10 µm. (E) HEK GFP-LC3 cells were transfected with myc-SNX18 WT or myc-SNX18 PX-BAR constructs, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3. (F) HeLa GFP-LC3 cells were transfected with siRNA against Atg16L1 and later with myc-SNX18 constructs before immunostaining and confocal imaging. Bar, 5 µm.