EMBO/soda-nel huggingface dataset:
SLC6A20 transporter: a novel regulator of brain glycine homeostasis and NMDAR function (2020)
Figure 6

Figure 6. Slc6a20a-mutant mice show abnormal increases in extracellular glycine levels and NMDAR function.

(A) Schematic diagram showing the generation of Slc6a20a-mutant mice. The SLC6A20 antibody is denoted by "Y" above exon (Ex) 6.

(B) Decreased levels of Slc6a20a, but not Slc6a20b, mRNAs in the brain of Slc6a20a+/- (HT) and Slc6a20a-/- (KO) mice (P21-28), as shown by qRT-PCR analysis. (n = 4, 3, 3 mice for WT, HT, and KO; *P < 0.05, **P < 0.01, ns, not significant, one-way ANOVA with Bonferroni's test). The error bars represent SEM.

(C) Reduced expression of SLC6A20 proteins in the brains of Slc6a20a+/- and Slc6a20a-/- mice (P21 for HT and P24-28 for WT and KO), as indicated by immunoblot analysis of total brain lysates. β-actin and α-tubulin were used as loading controls. (n = 3 mice for WT, HT, and KO, *P < 0.05, Student's t-test). The error bars represent SEM.

(D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM.

(E) Increased NMDA/AMPA ratio at hippocampal SC-CA1 synapses in Slc6a20a+/- mice (P17-20), as indicated by the ratio of NMDAR-EPSCs to AMPAR-EPSCs. (n = 9 neurons from 3 mice for WT and 11 (4) for Slc6a20a HT mice, **P < 0.01, Mann Whitney U test). The error bars represent SEM.

(F) Normal frequency and amplitude of mEPSCs in hippocampal CA1 pyramidal cells in Slc6a20a+/- mice (P17-20). (mEPSC, n = 14 neurons from 3 mice for WT and 16 (3) for HT; ns, not significant, Student's t-test). The error bars represent SEM.
The outer membrane protein MDI promotes local protein synthesis and mtDNA replication in the Drosophila ovary (2016)
Figure 1

Figure 1. mdi gene, protein and mdi1 deletion.

A. Schematic drawing of the mdi genomic locus showing the CG3249 (mdi) transcript (gray box) and coding region (black box), and the location of the gfp insertion generated by CRISPR/ Cas9-mediated recombination with 2 guide RNAs (arrows). The resulting fusion protein is referred to as MDI-GFP. The mdi1 mutation is a 2.4 kb deletion that removes most of the mdi coding region.

B. Western blots of MDI protein in wild type (wt) and mdi1 flies, showing that mdi1 is a protein-null mutation. Tubulin was used as loading control.

C. Ovarioles expressing MDI-GFP stained with a mitochondrial marker, ATP synthase (ATP-S). Overlapping signals (merge) indicate that MDI localizes to mitochondria. Note that MDI is highly expressed in germ cells at late germarium stages (left panels) and in egg chambers (right panels). Bars, 10 m.
The outer membrane protein MDI promotes local protein synthesis and mtDNA replication in the Drosophila ovary (2016)
Figure 2

Figure 2. MDI promotes mtDNA replication in the ovary and is essential for female fertility.

A-C. mtDNA replication in wt (A) and mdi1ovarioles (B, C) as illustrated by EdU incorporation. Arrows point to mitochondrial DNA, arrowheads to nuclei. In wt ovaries (A), mtDNA replication starts at germarium stage 2B and lasts into egg-chamber stage 2 (S2). In most mdi1 ovarioles (B), mtDNA replication is undetectable at these stages, whereas in some (C) it appears delayed until germarium stage 3. Bars, 10 m.

D. Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls. Each data point represents the mean of 3 independent replicates. Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was determined as the average of 3 biological repeats. P values of comparing mdi1; nos> mdi to mdi1: hatching rate, P= 1.1995E-05; mtDNA, P= 0.0078. P values of comparing mdi1; nos> hAKAP1 to mdi1: hatching rate, P= 1.6998E-05; mtDNA level, P= 0.0009.
The outer membrane protein MDI promotes local protein synthesis and mtDNA replication in the Drosophila ovary (2016)
Figure 3

Figure 3. Functional-genetic analyses of MDI and its interaction with Larp.

A. Schematic drawing of MDI protein and deletion mutants. These proteins were expressed under the nanos-gal4 driver, and tested for their ability rescue the hatching rate and mtDNA level of eggs from mdi1 females (progeny column) or for localization and recruitment of partner protein Larp to mitochondria in cultured cells (localization column). N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was the average of 3 biological repeats.

B. Representative images of S2 cells expressing GFP fusions (green) of the MDI proteins diagramed in (A). Staining with MitoTracker Red (red) to label mitochondria shows an overlapping signal (yellow), indicating proper localization to mitochondria for all fusion proteins except MDI∆MTS.

C. Submitochondrial localization of MDI. Mitochondria from cultured cells expressing an MDI-myc fusion protein were kept intact or subjected to swelling to disrupt their outer membranes, and digested or not with Protease K (PK). Protein extracts were subjected to western blot analysis. Tom20, cytochrome C (cyt.C) and SOD2 were used as markers of the outer membrane, inter-membrane space and matrix, respectively. MDI behaved like an outer-membrane protein.

D. Western blots of MDI protein in wt and mdi knockout (mdi-ko) cells confirming the mdi-ko cells completely lacked MDI protein. Actin was used as loading control.

E. Representative images of wt or mdi-ko cells expressing a Larp-GFP fusion protein and stained with MitoTracker Red to label mitochondria. The majority of Larp localized to mitochondria in wt cells (overlapping red and green signal), but diffused into the cytoplasm of mdi-ko cells.

F. Representative images of mdi-ko cells co-expressing Larp-GFP with MDI or the deletion mutants diagramed in (A) fused to the mCherry. Of the 4 MDI deletions that localized to mitochondria, 2 (MDIΔR and MDIΔTudor) did not allow Larp-GFP to localize to mitochondria. The localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants, and the hatching rate and mtDNA level in their eggs were determined. The result was summarized in (A).

Bars in (B), (E), (F), 10 m.
The outer membrane protein MDI promotes local protein synthesis and mtDNA replication in the Drosophila ovary (2016)
Figure 4

Figure 4. MDI recruits Larp to mitochondria.

A. Co-immunoprecipitation of Larp with MDI-myc in transfected S2 cells. Tubulin was used as loading control.

B. mtDNA replication, as illustrated by EdU incorporation, in wt and larp mutant (larp) ovarioles. mtDNA replication is dramatically reduced in the larp ovariole. Arrows: mtDNA; arrowheads: nuclei.

C. wt and mdi1 egg chambers stained for Larp (green) and ATP-S (red) to reveal mitochondria. Larp closely associates with mitochondria in wt egg chambers. Mitochondria in mdi1 flies are clumped together, and completely lack Larp staining. Bars in (B) & (C): 10 m.
The outer membrane protein MDI promotes local protein synthesis and mtDNA replication in the Drosophila ovary (2016)
Figure 5

Figure 5. Protein synthesis on the mitochondrial surface.

A. Nascent protein synthesis revealed by HpG incorporation (green) in wt and mdi1 egg chambers. Arrowheads point to the HpG signal on the ER in the perinuclear region and cell periphery. Arrows point to HpG signal associated with mitochondria. Mitochondria are marked by a Tom20-mCherry (red) created by inserting mCherry at the endogenous Tom20 locus.

B. ER location is unaltered in mdi1 egg chambers. An ER marker (ER-GFP) was expressed in wt and mdi1 egg chambers that were co-stained with ATP-S to mark mitochondria. ER localizes to the perinuclear region, cytoplasm and cell periphery in both wt and mdi1 egg chambers.

Bars in (A) & (B), 10 m.
The outer membrane protein MDI promotes local protein synthesis and mtDNA replication in the Drosophila ovary (2016)
Figure 6

Figure 6. MDI-Larp complex promotes the de novo synthesis or import of a subset of nuclear-encoded mitochondrial proteins.

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. A. Nascent protein synthesis was monitored by AHA incorporation, and detected by anti-biotin antibody. Tom20 served as a loading control. Note that the synthesis of nuclear-encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20-Larp (mdi1/TL).

B. Western blots of mitochondrial proteins in ovarioles of wt, mdi1 and mdi1 flies expressing Tom20-Larp (mdi1/TL) fusion protein. Note that levels of Tamas, TFAM, mRpL19 and COX4 were reduced in mdi1 flies, but restored in mdi1/TL flies.

C. Nascent protein synthesis of Tamas and TFAM was decreased in mdi1 ovary, whereas mtSSB was not affected. Tfamgfp, Tamasgfp and mtSSBgfp were expressed in wt or mdi1 background. Nascent proteins were labeled by AHA incorporation and then the GFP-tagged protein was immunopurified with a GFP antibody and the nascent protein synthesis was detected by anti-biotin antibody.

D. Representative profile of 254 nm absorbance of wt, mdi1 and mdi1/TL ovary extracts.

E. Polysome mRNA profiling for tamas, tfam, mtSSB, cox4 and mRPL19 in wt, mdi1 and mdi1/TL ovary. The percentage of mRNA for each gene in non-polysomal fractions (N.P, including ribosomal subunits and monosome-associated) and polysomal fractions (poly) was calculated and plotted. The fractions of tamas, tfam, cox4 and mRPL19 mRNAs in the polysomal fractions were significantly decreased in mdi1 compared to wt, but were restored in mdi1/TL flies. N=4 for all samples. P values of comparing wt to mdi1: tamas, P= 0.0055; tfam, P= 0.001; cox4, P=0.0066; mRPL19, P=0.0097. P values of comparing mdi1/TL to mdi1: tamas, P= 0.0206; tfam, P= 0.0346; cox4, P=0.0036; mRPL19, P=0.0016.

F. The nascent protein synthesis of COX4 and mRPL19 were decreased in mdi1 ovary and were restored mdi1/TL flies. The proteins were immunopurified with antibodies against the endogenous proteins.
The outer membrane protein MDI promotes local protein synthesis and mtDNA replication in the Drosophila ovary (2016)
Figure 7

Figure 7. Targeting Larp to the mitochondrial surface partially rescues the mdi1 phenotype.

A. Mitochondrial localization of a Tom20-LarpGFP (Tom20-Larp) fusion protein in an mdi1 egg chamber stained with ATP-S to reveal mitochondria. Bars, 20 m.

B. Hatching rates and mtDNA contents of eggs produced by flies with different genotypes relative to wt control; each data point represents the mean of 3 independent replicates. Bars, std. Expression of Tom20-Larp in mdi1 (mdi1;Tom20-Larp) significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was the average of 3 biological repeats. P values of comparing mdi1;Tom20-Larp to mdi1: for mtDNA level, P=0.0209; for hatching rate, P=3.4758E-05.

C. Model of the role of MDI and Larp in mitochondrial biogenesis. MDI (green rectangle) localizes at the mitochondrial outer membrane, and recruits Larp (blue rectangle) to the mitochondrial surface. Larp interacts with polysome (blobs) and translation stimulators, and stimulate the translation of a subset of nuclear-encoded mRNAs. Proteins synthesized on the mitochondrial surface are in close vicinity of the Tom-Tim mitochondrial transporter complexes, which would facilitate their rapid transport into the matrix. The targets of MDI-Larp complexes include most nuclear-encoded ETC subunits, mitochondrial ribosomal proteins, TFAM, and mtDNA polymerase (Tamas) (collectively represented in red). Mitochondrial ribosomes are required for the biogenesis of proteins encoded by mtDNA, all of which are ETC subunits. Thus MDI-Larp complexes seem to coordinate the expression of both nuclear and mitochondrial genome to promote the biogenesis of ETC complexes.
Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome (2017)
Figure 2

Figure 2. PPP1R13L SeVa and expression.

A. PPP1R13L mRNA expression level is lower in VI10 PDFs (set as 1) than in CFs. B. and C. Western blots (WBs) analyses demonstrated the absence of iASPP in VI10, VI4 and VII1 PDFs. D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band.
Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome (2017)
Figure 3

Figure 3. Upregulation of pro-inflammatory mediators in PDFs.

A. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs than in CFs. The expression level in resting conditions of the PDFs was set as 1. Differences between knockdown and control with p values ≤ 0.05 (Student’s t test) are indicated with an asterisk. B. as in A. for patient VII1. C. knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators. Differences between knockdown and control with p values (Student’s t test) are indicated with asterisks in the graph.
Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome (2017)
Figure 4

Figure 4. NF-kB-dependent upregulation of pro-inflammatory cytokines.

A. WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs. B. ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS-stimulated PDFs than in LPS-stimulated-CFs. The binding activity at the IL1B promoter was set as 1. Differences between knockdown and control with p value= 0.00436 (Student’s t test) are indicated with an asterisk. C. NF-kB inhibitor BOT-64 blocked the inducible mRNA expression of the inflammatory cytokines. Differences between knockdown and control with p values (Student’s t test) are indicated with an asterisks and exact values.
Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome (2017)
Figure 5

Figure 5. iASPP regulates the innate immune response of murine neonatal cardiomyocytes.

A. WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes. B. qPCR showed that ppp1r13l-knocked down murine cardiomyocytes (using two alternative shRNA sequences) expressed higher level of pro-inflammatory cytokine mRNAs after 4 hrs of LPS stimulation. The expression in ppp1r13l-knocked down murine cardiomyocytes was set as 1. Genome wide expression analysis is presented in Figure 1S. Differences between knockdown and control with p values (Student’s t test) are indicated with asterisks and exact values.
Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome (2017)
Figure 6

Figure 6. Hyper cardiac-inflammatory response of ppp1r13l-deficient mice.

A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C (Mybpc3), phospholamban (Pln), ryanodine receptor 2 (Ryr2), and sarcoglycan gamma (Sgcg). Hearts removed from perfused mice and mRNA was subjected for q PCR. Difference between knockdown and control with p values = 0.03 (Student’s t test) is indicated with an asterisk. B. A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn, 7-week- and 12-week- hearts (p<0.05 threshold, calculated by Deseq algorithm “R”). The expression level of s100a9 was higher in wa3 mice than in WT by 2.8 times. For the full list of genes see Tables S1 and S2, and functional analyses in Figures S3-S6. C. Hierarchical clustering heat map of differentially expressed genes in the hearts of 7- and 12- week old wa3 mice in comparison to WT.
Sequence variation in PPP1R13L results in a novel form of cardio-cutaneous syndrome (2017)
Figure 7

Figure 7. Ppp1r13l-knockout mice exhibits cardiac hypersensitivity to inflammatory triggers.

A. LPS (5mg/Kg for 2 hrs) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts. The results are presented as fold induction of untreated controls. The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts, while of Mybpc3 and Sgcg was unchanged. For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7. Differences between knockdown and control with p values =0.04998 (Student’s t test) are indicated with an asterisk.

B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS (1mg/Kg).
TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes (2022)
Figure 1

Figure 1. Stm1 binds to vacant 80S ribosome upon TORC1 inhibition.

A. Heat map showing the fraction distribution of the top 25 most abundant proteins across the polysome profiles treated with or without rapamycin for 1 hour. The proteins are sorted according to their distribution in the 80S fraction (fraction 6) upon rapamycin treatment (Fraction number 6). Individual polysome profiles were separated on a 5-50% sucrose density gradient containing 50 mM KCl into 14 fractions, and fraction numbers 3 to 13 were analyzed by mass spectrometry.

B. Distribution of proteins Stm1, Rps14a, and Rpl6b in the fractions across the polysome profile obtained with or without rapamycin treatment for 1 hour as identified by MS/MS.

C-F. Polysome profiling of yeast cells with (C-E) or without (F) cycloheximide (CHX) treatment, immunoblots for Stm1 and Rpl3 in polysome fractions, and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient-rich condition (C, F), rapamycin treatment for 1 hour (D), or nitrogen starvation for 1 hour (E). Individual polysome profiles were fractionated on a 5-50% sucrose density gradient containing 150 mM KCl into 9 fractions, precipitated and immunoblotted for Stm1 and Rpl3. Rpl3 is used as a loading control. Each experiment was repeated at least three times. Data are presented as the mean ± SD.
TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes (2022)
Figure 2

Figure 2. Stm1 forms vacant 80S ribosomes upon TORC1 inhibition.

A-D. Polysome profiles of wild-type (wt) and stm1∆ cells with (A-C) or without (D) CHX treatment under nutrient-rich conditions (A, D), with rapamycin treatment for 1 hour (B), or under nitrogen starvation for 1 hour (C) using 5-50% sucrose density gradients containing 150 mM KCl. At least three biological replicates are analyzed for each condition.

E-F. Polysome profiles of formaldehyde cross-linked wild-type (wt) and stm1∆ cells using 5-50% sucrose density gradients containing 150 mM KCl (E) or 300 mM KCl (F).

G. Polysome profiles of stm1∆ cells containing either full-length or N-terminally deleted or C-terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour.

H. Binding of full-length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E. 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3. At least two biological replicates are used for each mutant.
TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes (2022)
Figure 3

Figure 3. TORC1 phosphorylates and inhibits Stm1.

A-B. In vitro kinase assay (A) and its quantitation (B) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P-γ-ATP. Immunoblots for Stm1 and Raptor are shown. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used as an inhibitor of mTOR. Minimum of three replicates are used for statistical analysis by unpaired t-test. Data are presented as the mean ± SD.

C. Polysome profiles of wild-type and stm1∆ cells under nitrogen starvation for 1 hour.

D. Polysome profiles of wild-type and STM1AAA cells under nitrogen starvation for 1 hour.

E. Polysome profiles of wild-type and STM1EEE cells under nitrogen starvation for 1 hour.

F-H. Immunoblots for Stm1 and Rpl3 from the ribosomal fractions obtained from wild-type (F), STM1AAA (G), and STM1EEE (H) cells under nitrogen starvation for 1 hour.
TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes (2022)
Figure 4

Figure 4. Stm1 protects ribosomes from proteasomal degradation.

A-B. Ribosomal subunit profiles (A) and quantitation of ribosome content (B) of stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. For the ribosomal subunit profile analyses, the cell extracts were treated with 50 mM EDTA and separated on 5-25% sucrose density gradients. At least five biological replicates are used for calculation of p-values from multiple unpaired t-tests are indicated in the graphs.

C-D. Immunoblot (C) and the quantification (D) of Rpl3 in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.

E-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).

H-I. Immunoblot for ubiquitin in the supernatant and ribosomal pellet (H) obtained from stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. Immunoblots for Stm1 and Rpl3 are shown as controls. Quantification of ribosomal ubiquitylation (I) relative to wild-type is shown. The ribosome pellets were obtained using a 1 M sucrose cushion. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.

J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.

Data information: The respective p-values are indicated in the figure. Data are presented as the mean ± SD.
TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes (2022)
Figure 5

Figure 5. TORC1 activates dormant ribosomes by inhibiting Stm1.

A-B. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.

C. Germination efficiency of spores upon dissection of tetrads (schematic in upper panel) from diploid wild-type and stm1∆ cells on YPD agar media after 6 hours of incubation. Minimum of four replicates are used for unpaired t-test.

D-E. Representative fluorescent microscopic images for expression of Rpl24-GFP (D) and its intensity quantification (E) in tetrads of control (RPL24GFP) and stm1∆ RPL24GFP cells (n=100, each). Experiments are performed at least three times. Statistical analysis is done by unpaired t-test.

Data information: Data are presented as the mean ± SD.