EMBO/soda-nel huggingface dataset:
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 2

Figure 2. The BH3 region and the groove are partially embedded in the MOM.

A. Top-left panel, structure of the BH3-in-groove dimer (PDB entry 4BDU) with the predicted MOM-engaging residues presented in stick form. Bottom-left panel, structure of the BH3-in-groove dimer with the predicted protein-buried residues presented in stick form. Top-right panel, structure of the BH3-in-groove dimer without helix α5 (modified from PDB entry 4BDU), and bottom-right panel, structure of α5 (extracted from the NMR structure of Bax monomer, PDB entry 1F16). In both structures in the right panels, the residues that are buried in the MOM or exposed to the aqueous milieu as the IASD labeling data in (B-D) confirmed are presented in yellow or cyan stick form, respectively. In all panels, the color codes for the two monomers are the same as that in Figure 1A, and indicated.

B. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in helix α2, α3, α4 or α5, were activated and targeted to the mitochondria. The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS, urea, or both. After 30 min, the labeling reactions were stopped by mercaptoethanol. For the “0 min” controls, the samples were pretreated with mercaptoethanol before addition of IASD. The IASD-labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing (IEF; as indicated) or gradient SDS-PAGE and detected by phosphor-imaging. Triangles and arrows indicate the unlabeled and IASD-labeled Bax proteins, respectively. n = 3 for V111C, and K119C; 4 for W107C, and A117C; 2 for other mutants.

C-D. The phosphor-imaging data for IASD labeling of Bax mutants in (B) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3. The membrane burial indices were normalized by that of G179C in α9, a reference for the membrane-buried residues. The resulting relative membrane burial indices are shown in (C). The residues with the relative membrane burial index ≥ 40% of that of Gly179 are considered as buried in the MOM. Similarly, the relative protein burial indices shown in (D) were obtained by using Y115C in α5 as a reference for the protein-buried residues. The residues with the relative protein burial index ≥ 40% of that of Tyr115 are considered as buried in the protein or its complex.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 3

Figure 3. The BH3-in-groove dimer interface rearranges to form a helices α2-α3-α4 dimer interface.

A-B. Oxidized mitochondria with the radioactive single- or double-cysteine Bax proteins, or the single-cysteine Bax protein pairs were prepared and analyzed as in Figure 1B.

C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D.

D. The best structural model for the helices α2-α3-α4 dimer is shown with the residue pairs replaced by cysteine pairs in (B) presented as in Figure 1A. The β carbon atoms of each residue pair are linked by a dashed line with the distance ranging from 4.7 to 5.2 Å.

E. The structural model for the α2-α3-α4 dimer is shown with the residues that are buried in the MOM or exposed to the aqueous milieu as determined in Figure 2 presented in yellow or cyan stick form, respectively. The pdb file for the model is provided as Dataset EV1 and named as “Helices a2-a3-a4 dimer.pdb”.

In (D-E), the color codes for the two monomers are indicated. In (A-C), protein standards, Bax monomers, and disulfide-linked Bax dimers are indicated as in Figure 1B. In some targeting reactions, the mitochondria were pretreated with NEM (NEM-mito), while in others the mitochondria were untreated (mito). In (A-B), n = 2 for C62,S72C, R94C, and L59C+L76C; 3 for S72C, S55C, and S55C+R94C; 4 for C62; 6 for E69C, L59C, and L76C; 8 for L59C,L76C. In (C), n = 2 for each mutant.

The triangle-indicated bands generated from the untreated mitochondria in (A) are disulfide-linked heterodimers between the Bax cysteine mutants and unknown mitochondrial proteins, as they disappeared if the mitochondria were pretreated with NEM to block the sulfhydryls of the mitochondrial proteins before adding the Bax mutants (e.g., compare the products from E69C at the untreated mitochondria in lane 8 of Figure 3A to those from the same mutant at the NEM-pretreated mitochondria in lane 4 of Figure 3C, or the products from S72C at the untreated mitochondria in lane 4 of Figure 3A to those from the same mutant at the NEM-pretreated mitochondria in lane 4 of Figure EV4A). In contrast, the disulfide-linked Bax homodimers were still formed in the NEM-pretreated mitochondria (e.g., the arrow-marked band in lane 4 of Figure 3C and lane 4 of Figure EV4A).
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 4

Figure 4. G108E mutation in the groove disrupts the BH3-in-groove as well as the helices α2-α3-α4 Bax homodimer interface, but not tBid-Bax heterodimer interface.

A. Left panel, part of the BH3-in-groove dimer structure is shown with Gly108 from one monomer presented in sphere form, and Gly67 and Leu70 from the other monomer that have van der Waals contacts with the Gly108 presented in stick form. Asp68 and Asp71 from the other monomer are also presented in stick form. Right panel, the same structure is shown with the Gly108 changed to glutamate (G108E), which results in steric clashes with the Gly67, Leu70 and Asp71, and electrostatic repulsions with the Asp68 and Asp71.

B and D. The radioactive single- or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated (mito) or pretreated with NEM (NEM-mito), then oxidized and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.

C. The in vitro synthesized [35S]Met-labeled Bax E69C protein with or without the additional mutations (G108E and/or S184V) were activated by the in vitro synthesized [35S]Met-labeled tBid L105C protein and targeted to the Bax-/-/Bak-/- mitochondria that were pretreated with NEM. The resulting mitochondria were isolated and oxidized by CuPhe for 0 or 30 min. The resulting “0 min” samples (1 equivalent each) and “30 min” samples (1 equivalent each) were analyzed by non-reducing SDS-PAGE and phosphor-imaging. The remaining “30 min” samples (4 equivalent each) were immunoprecipitated (IP) by either Bax- or tBid-specific antibody, and then analyzed by non-reducing SDS-PAGE and phosphor-imaging. The identities of the four major products, indicated on the right side of the image, were based on their Mr and recognition by the respective antibody. n = 2.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 5

Figure 5. Helix α9 is partially embedded in the MOM.

A. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in α9, were activated and targeted to the mitochondria, and labeled with IASD as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using gradient SDS-PAGE, detected by phosphor-imaging and indicated by triangles and arrows, respectively. For all mutants, n = 2, except for A183C, n = 3.

B-C. The IASD labeling data in (A) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5, from which the relative membrane (B) or protein (C) burial indices were obtained as described in Figure 2C-D, and shown.

D. The structure of α9 (extracted from the NMR structure of Bax monomer, PDB entry 1F16) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu, as the IASD labeling data in (A-C) suggested, presented in yellow or cyan stick form, respectively.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 6

Figure 6. Helix α9 homodimerizes with two interfacial conformations.

A and F. Structural model for intersected (A) or parallel (F) α9 homodimer is shown with one monomer colored brown (α9) and the other gray (α9’). The residue pairs that formed disulfide bonds after they were replaced with cysteine pairs in (B) or (D), respectively, are presented in stick form, and their β carbon atoms linked by dashed lines with the distances indicated in Å, except that the Gly179 pair is linked by a dashed line via their α carbon atoms with the distance indicated. The pdb files for the models are provided as Datasets EV2 and EV3 and named as “Helix a9 intersected dimer.pdb” and “Helix a9 parallel dimer.pdb”.

B and D. The oxidized mitochondria with radioactive single-cysteine Bax proteins were prepared and analyzed as in Figure 1B.

C and E. The oxidized mitochondria with the radioactive single-cysteine Bax proteins were prepared, subjected to Na2CO3 extraction, and analyzed as in Figure 1D.

In (B-E), protein standards, Bax monomers and disulfide-linked Bax dimers are indicated as in Figure 1B. n = 3 for A183C, and A178C; 2 for other mutants.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 7

Figure 7. G179I or T182I mutation in α9 disrupts the α9 dimer but not the BH3-in-groove and α2-α3-α4 dimers, whereas G108E mutation in the groove disrupts the α9 dimer.

A. Intersected α9 dimer: the structural model on the left depicts one monomer with Gly179 in sphere form, and the other monomer with Phe176, Gly179 and Val180 that have van der Waals’ contacts with the first Gly179 in stick form. The same model is shown on the right, except that the two Gly179 are changed to isoleucine (G179I), which results in steric clashes between the Ile179 in one monomer and the Phe176, Ile179 and Val180 in the other monomer, and vice versa.

Parallel α9 dimer: the structural model on the left depicts the two interfacial Thr182 that have van der Waals’ contacts in sphere or stick form. The same model is shown on the right, except that the two Thr182 are changed to isoleucine (T182I), which results in a steric clash with each other.

B-D. The in vitro synthesized radioactive single or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria, oxidized, and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 8

Figure 8. Combinations of either the BH3-in-groove or the α2-α3-α4 dimer interface with the α9 dimer interface mediate Bax oligomerization.

A. The oxidized mitochondria with radioactive single, double, or triple-cysteine Bax proteins were prepared and analyzed by non-reducing or reducing SDS-PAGE as in Figure 1B. Protein standards, Bax monomers and disulfide-linked Bax homodimers are also indicated as in Figure 1B. Closed triangles indicate disulfide-linked higher order Bax homo-oligomers. Open triangles indicate a background band that could not be reduced, and hence is not a disulfide-linked complex.

B. The BMH-treated mitochondria with the radioactive single- and double-cysteine Bax proteins were prepared and analyzed as in Figure EV3G, except that closed triangles indicate the BMH-linked Bax oligomers.

In (A-B), n = 2 for all mutants.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 9

Figure 9. The BH3-in-groove dimerization is required for Bax to permeabilize the MOM.

A-C. The single or double-cysteine Bax proteins with or without the indicated mutations were synthesized in vitro, activated by tBid protein and targeted to the Bax-/-/Bak-/- mitochondria. Cytochrome c release from the mitochondria was measured using ELISA. The dots are the fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B, and the lines are the means. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.
BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes (2015)
Figure 10

Figure 10. The α9 dimerization is not required for the release of cytochrome c from mitochondria, but facilitates the release of Smac and larger proteins.

A. The cytochrome c release by the indicated Bax mutants was measured in two independent replicates, corrected and shown as in Figure 9. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.

B. Purified wild-type (WT) or mutant (G179I or T182I) Bax protein of the indicated concentration was incubated with the Bax-/-/Bak-/- mitochondria in the absence or presence of tBid protein. The proteins released from and those remaining in the mitochondria were separated and analyzed by SDS-PAGE and immunoblotting with antibody specific to either cytochrome c or Smac. n = 2.

C. Purified wild-type or mutant Bax protein of the indicated concentration alone or together with purified cBid was incubated with the Bax-/-/Bak-/- mitochondria containing Smac-mCherry protein. Release of Smac-mCherry was detected by measuring the fluorescence in the supernatant after the mitochondria were pelleted by centrifugation. The dots are the fractions of Smac-mCherry release from three independent replicates, and the lines are the means.

D. The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein, and then labeled with IASD, as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using either gradient SDS-PAGE or IEF, detected by phosphor-imaging, and indicated by triangles and arrows, respectively. n = 3 for A178C,G179I; 2 for other mutants.

E. The IASD labeling data in (D) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A, from which the relative membrane burial indices were obtained as described in Figure 2C, and shown.
Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice (2022)
Figure 3

Figure 3. Landscape of neuron-type-specific lincRNAs in somatosensory neurons by Smart-seq2 and 10x Genomics.

(A) Heatmap showing the neuron-type-specific lincRNAs consistently detected in two scRNA-seq methods. Rows represent specific cell types. Columns represent individual lincRNAs.

(B) Venn diagram showing the overlapping of highly neuron-type-specific lincRNAs (SPMcluster ≥ 0.8 and TPMcluster ≥ 1) between two scRNA-seq methods.

(C) Composition of 200 highly neuron-type-specific lincRNAs in distinct neuron types.

(D) Characterization of highly neuron-type-specific lincRNAs in two scRNA-seq methods, ranked by evaluation of robust rank aggregation (RRA) method.

(E) Representative images (Left) of smFISH in DRG section, tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that C530044C16Rik was co-localized with Gal-Cre; Ai9 tdTomato neurons (n = 7), but not with Sst-Cre neurons (n = 6).

(F) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that CLAP was co-localized with Sst-Cre; Ai9 tdTomato neurons (n = 6), but not with Gal-Cre neurons (n = 29).

(G) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that Gm11549 was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 5), but not with Mrgprd-Cre neurons (n = 6).

(H) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that 2310002F09Rik was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 6), but not with Slc17a8-Cre neurons (n = 6).

(I-K) Representative images (Right) of double smFISH showed that 2310002F09Rik was rarely co-localized with C530044C16Rik (I), CLAP (J) and Gm11549 (K). Scale bar, 10 µm.

Data information: (E, F, G, H) The results are presented as mean ± SEM. n indicates the number of tissue sections at least from 3 mice.
Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice (2022)
Figure 4

Figure 4. Coexpression of neuron-type-specific lincRNAs with neighboring mRNAs.

(A) Dot plot showed that the highly neuron-type-specific lincRNAs and their adjacent protein-coding genes were more likely to predominantly express in the same neuron type by both Smart-seq2 and 10x Genomics.

(B) Statistical results showed that the percentage of consistent lincRNA-mRNA pairs were higher than those non-consistent pairs in both Smart-seq2 and 10x Genomics. Top-ranking list of lincRNA-mRNA pairs is showing at right.

(C) scRNA-seq coverage showed that C3-type-specific AK043662 is genomically adjacent to Wfdc2 (Upper) and C5-type-specific 2310002F09Rik is genomically adjacent to Klk8 (Bottom).

(D) Representative smFISH images in DRG sections and quantitative results showed that 2310002F09Rik and Gm14273 were more enriched in the nucleus, whereas CLAP and Gm11549 were mainly distributed in the cytoplasm. DAPI served as a nuclear marker. Scale bar, 10 μm.
Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice (2022)
Figure 5

Figure 5. CLAP is highly conserved and abundantly expressed in C2 type of DRG neurons.

(A) Boxplot showing the abundance of highly expressed neuron-type-specific lincRNAs in Smart-seq2 and 10x Genomics. Each dot represents a lincRNA, colored (Red) by its conservation between mouse and human. In boxplot, central band, boxes and whiskers represent median value, second/third quartiles and minimum/maxmun value to second/third quartiles, respectively.

(B) Sequence conservation analysis of lincRNA CLAP and upstream mRNA Pirt in 9 different species including mouse, human, chimpanzee, rhesus monkey, rat, cow, dog, chicken and zebrafish, which were visualized with the VISTA browser. CDS, coding sequence. UTR, untranslated region.

(C) Gene co-expression network identified by WGCNA shows that CLAP co-expressed with known marker genes such as Sst and Nppb in C2-type neurons. Blue and red colors represent mRNAs and lincRNAs, respectively.

(D) qPCR showed that CLAP was highly expressed in the DRG and other visceral organs such as small intestine, stomach and lung. The results are presented as mean ± SEM (n = 3).

(E) qPCR showed that the expression of CLAP was gradually increased from E13.5 to adult DRGs. The results are presented as mean ± SEM (n = 3).

(F) Representative bright-field and fluorescent images showing the Sst-cre; Ai9 neurons for calcium imaging following siCLAP transfection. Scale bar, 10 μm.

(G) qPCR showed that the expression of CLAP was decreased by siRNA treatment in cultured Sst-Cre; Ai9 DRG neurons. The results are presented as mean ± SEM (n = 3). ** P < 0.01 versus siNC.

(H) Traces showed that a Sst-Cre; Ai9 tdTomato neuron was evoked by histamine (1 mM), 5-HT (100 μM), chloroquine (1 mM), capsaicin (1 μM) and KCl (80 mM) in the calcium imaging.

(I-L) The percentage of Sst-Cre; Ai9 neurons evoked by histamine (I) was decreased by knockdown of CLAP, whereas the percentage of these neurons affected by 5-HT (J), chloroquine (K), capsaicin (L) and KCl (M) were not changed. The results are presented as mean ± SEM (n = 4). ** P < 0.01 versus siNC.

Data information: (G, I, J, K L and M) Unpaired T-Test.
Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice (2022)
Figure 6

Figure 6. CLAP is sufficient for histamine-induced scratching

(A) Research flowchart for acute itch behavioral test following intrathecal injection of siRNA in mice. Before acute itch behavior testing, mice were intrathecally injected with siRNA 2 times in 3 d.

(B) qPCR showed that the CLAP level was significantly decreased by siCLAP in the DRG following intrathecal injection. The results are presented as mean ± SEM (siNC = 12, siCLAP = 15). *** P < 0.001 versus siNC.

(C-E) Knockdown of CLAP reduced itch scratching evoked by histamine (C), but not 5-HT (D) and chloroquine (E). The results are presented as mean ± SEM (For histamine and 5-HT, siNC = 12，siCLAP = 15; for chloroquine, siNC = 5，siCLAP = 8 ). ** P < 0.01 versus siNC.

(F) Research flowchart for AAV-mediated knockdown of CLAP by intrathecal injection of AAV-shCLAP-GFP.

(G) qPCR showed that the CLAP level was significantly decreased by AAV-shCLAP-GFP in DRGs following intrathecal injection and testing of acute itch behavior. The results are presented as mean ± SEM (shNC, n = 8; shCLAP, n = 7). *** P < 0.001 versus shNC.

(H-J) Knockdown of CLAP reduced the scratching induced by histamine (H), but not that by 5-HT (I) and chloroquine (J). The results are presented as mean ± SEM (shNC, n = 9; shCLAP, n = 8). * P < 0.05 versus shNC.

(K) Research flowchart for AAV-mediated overexpression of CLAP by intrathecal injection of AAV-CLAP.

(L) qPCR showed that the CLAP level was significantly increased by AAV-CLAP in DRGs following intrathecal injection. The results are presented as mean ± SEM (n = 12). ** P < 0.05 versus vector.

(M and N) Overexpression of CLAP increase the scratching induced by histamine (K), but not that by chloroquine (L). The results are presented as mean ± SEM (n = 12). * P < 0.05 versus vector.

(O) Flowchart for mechanical, hot and cold behavioral tests following intrathecal injection of siRNA in mice.

(P-R) The threshold for mechanical (P), heat (Q) and cold (R) responses were not affected by knockdown of CLAP. The results are presented as mean ± SEM (siNC, n = 13; siCLAP, n = 7).

Data information: All statistical analysis were used Unpaired T-Test.