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The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

• Authors : Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael ...

• Source: eLife ; volume 5 ; ISSN 2050-084X

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Figure 12. Computational and experimental characterization of cancer-associated SETD8 mutants. ; (a) Cancer-associated mutations in the catalytic domain of SETD8 examined in this work. (b) Cartoon representations of TC with cancer-associated SETD8 mutations highlighted. (c) Differential residue-contact maps of cancer-associated SETD8 mutants in reference to wild-type apo-SETD8 (gray). Residue-residue contact map of wild-type apo-SETD8 is presented as a 162 × 162 matrix. The vertical and horizontal axes show the residue numbers of SETD8’s catalytic domain. The contact of a pair of residues is scored as ‘1’ if their distance is shorter than 4.0 Å; ‘0’ if the distance is equal or above 4.0 Å. For the 60 1ZKK(chain A)-seeded MD trajectory frames of wild-type apo-SETD8, the average contact fraction of each residue pair is presented in a square shape and depicted with a gray gradient at the corresponding vertical and horizontal coordinates. The contact fraction of cancer-associated SETD8 mutants were obtained in a similar manner. The vertical and horizontal coordinates of representative positive changes of the contact scores from wild-type to mutated SETD8 (newly acquired interactions) are highlighted in red-gradient squares with details expanded in the next panel. (d) Representative contacts in the differential residue-contact maps of cancer-associated SETD8 mutants. The contacts of SETD8 mutants with >3 fold gain of contact fraction relative to wild-type SETD8 are listed. Increased magnitude of the contact fraction is depicted in red gradient as described in the previous panel. Only positive changes (newly acquired interactions) are presented with the two residues involved labeled in left and top; the fold of the increase of their contact score labeled in bottom. (e) Cartoon representations of neo-conformations revealed by simulations of SETD8 mutants. Large conformational changes are observed in the SET-I (purple) and post-SET (orange) motifs. (f) Differential residue-contact maps of the structurally relaxed α-helix at the SET-I motif of SETD8 A296T mutant. Decrease of contact fraction relative to wild-type SETD8 is depicted in blue gradient. (g) Enzymatic activities of wild-type and mutated SETD8 determined by an in vitro radiometric assay with H4K20 peptide substrate. Here SETD8 mutants are categorized as the following: red, uncovered neo-conformations (Neo-conf.) with >90% loss of methyltransferase activity; green, populated inactive conformations (Pop. shift) with partially abolished methyltransferase activity; blue, no large change of differential contact maps with comparable methyltransferase activity with wild-type SETD8; brown, unknown relationship between differential contact maps and methyltransferase activities. Data are mean ±standard deviation (s.d.) of 3 replicates.

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Figure 6. Ribozyme self-synthesis and assembly of its own catalytic domain. ; (A) t5+1-catalysed syntheses of the five catalytic domain ‘+’ and ‘−’ segments via triplet extension of primers (grey) in −7˚C ice. Triplets are coloured by segment and shown alongside the lanes; longer oligonucleotide substrates (faded) were provided for α+ and ε+ syntheses to combat ribozyme-template pairing as shown in Figure 6—figure supplement 2 and Figure 6—figure supplement 3. The triplets were supplied at 5 μM (α+ to ε+), 10 μM (β− to ε−), or 20 μM (α−) each, with 0.5 μM primer/template (P/T; 1 μM for Fβ6/Tβ) and oligonucleotides equimolar to template sites. Use of substrates of more heterogenous compositions and lengths is shown in Figure 6—figure supplement 4 and Figure 6—figure supplement 5, respectively. Densitometry gave yields of full-length products (boxed, by % of total primer), and a geometric mean of the final extents of ligation across all 70 junctions in this self-synthesis context (78%). These segment sequences derive from t5b, a t5 variant with a neutral signature mutation (Supplementary file 1). (B) Secondary structure representation of a t5 catalytic domain (αβ+/γδε+, t5b sequence), formed via non-covalent assembly of t5+1-synthesised ‘+’ strand fragments in Figure 6—figure supplement 1, coloured by segment and synthesis substrate as in (A). (C) Activity of ribozyme-synthesised αβ+/γδε+ (B), compared to protein-synthesised αβ+/γδε+ and full-length t5b equivalents. These were assayed for synthesis of a δ- strand segment on a ribozyme-synthesised δ+ template, with added in vitro transcribed type 1 (2 μM each Rz, 5 μM triplets, 0.5 μM P/T, −7˚C 0.25× ice 10 days). The ribozyme-synthesized and assembled αβ+/γδε+ ribozyme is as active as in vitro transcribed equivalents, and can efficiently utilize ribozyme-synthesized RNA (δ+) as a template.

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Figure 1—figure supplement 2. Sequence alignment of worm ULP proteins. ; The alignment was performed by MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft/). The catalytic domain of ULP-4 is highlighted in red.

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Figure 3. HTT interacts with the C-terminal catalytic domain of PNKP. ; (A) Schematic illustrating various functional domains of PNKP expressed as FLAG-tagged peptides: (1) full-length PNKP containing N-terminal fork-head-associated (FHA) domain, central phosphatase (PHOS) and C-terminal kinase (KIN) domains; (2) FHA domain (1–130 amino acids); (3) PHOS domain (131–337 amino acids); (4) KIN domain (338–521 amino acids); (5) FHA and PHOS domains (1–137 amino acids); and (6) PHOS and KIN domains (131–521 amino acids). (B) Plasmids encoding full-length PNKP (FL-PNKP) or various PNKP domains were separately transfected into SH-SY5Y cells (lanes 1 to 6) and NEs isolated 48 hr post-transfection. Lanes 1 to 6 in the WB (upper panel) shows the pull-down of full-length PNKP (FL-PNKP) and various PNKP domains that were IP’d with an anti-FLAG Ab. The WB in the lower panel shows the presence of endogenous HTT (arrow) in the FLAG-IC. M: protein molecular weight marker. (C) Plasmids encoding full-length PNKP (FL-PNKP) and various PNKP domains were separately transfected into SH-SY5Y cells (lanes 1 to 6), NEs isolated, and HTT was IP’d with an anti-HTT antibody. The pull-down of endogenous HTT is shown in the upper panel (arrow). The HTT-IC was analyzed by WB (lower panel) to detect FL-PNKP or various PNKP domains with an anti-FLAG Ab (arrows). (D) Plasmids encoding FLAG-tagged full-length PNKP (FL-PNKP) or various PNKP domains were transfected separately into PC12 cells expressing full-length Myc-tagged normal HTT encoding 23Qs (Myc-FL-wtHTT-Q23) (lanes 1 to 6), NEs were isolated, and Myc-HTT IP’d with Myc tag antibody. Upper panel is the WB showing the IP of HTT with an anti-Myc tag antibody. The Myc-IC was analyzed by WB to assess interaction of various PNKP domains with HTT with an anti-FLAG antibody (lower panel, arrows). (E) Plasmids encoding the full-length PNKP (FL-PNKP) or various PNKP domains were transfected into PC12 cells expressing Myc-tagged full-length mutant HTT encoding 148Qs (Myc-FL-mHTT-Q148) (lanes 1 to 6), NEs isolated, and Myc-tagged HTT was IP’d with an anti-Myc-tag antibody. Upper panel is the WB showing IP of Myc-HTT with anti-Myc tag antibody. Interactions of FL-PNKP or various PNKP domains with FL-HTT were analyzed by WB with an anti-FLAG antibody (lower panel, arrows).

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Figure 4. The N-terminus of HTT interacts with the C-terminal catalytic domain of PNKP. ; (A) Schematic showing GFP-tagged N-terminal fragment of wild-type normal HTT encoding 23Qs or mutant HTT encoding 97Qs (NT-wtHTT-Q23 and NT-mHTT-Q97 plasmid vectors respectively; upper panel). SH-SY5Y cells were transfected with NT-wtHTT-Q23 or NT-mHTT-Q97, NEs isolated, fusion peptides IP’d from NE with an anti-GFP antibody, and WBs performed with respective antibodies to detect endogenous PNKP, ATXN3, or HTT in the GFP-IC (lower panel). (B) NEs from PC12 cells expressing Myc-tagged N-terminal fragment of wild-type normal HTT encoding 23Qs or mutant HTT encoding 148Qs (NT-wtHTT-Q23 or NT-mHTT-Q148, respectively) were isolated and the Myc-HTT was IP’d with an anti-Myc tag Ab and Myc-IC was analyzed by WBs to detect various TCR complex components with respective antibodies. (C) Plasmids encoding full-length PNKP (FL-PNKP) or various PNKP domains (lanes 1 to 6) were separately transfected into SH-SY5Y cells expressing the N-terminal fragment of HTT encoding 23Qs (Myc-NT-wtHTT-Q23), NEs were isolated and the NT-HTT was IP’d with an anti-Myc tag Ab. The upper panel shows pull down of Myc-NT-HTT-Q23. The Myc-IC was analyzed by WBs with an anti-FLAG Ab to detect FL-PNKP or PNKP domains (lower panel; arrows). (D) Plasmids encoding full-length PNKP (FL-PNKP) or various domains (lanes 1 to 6) were separately transfected into SH-SY5Y cells expressing the N-terminal fragment of mutant HTT encoding 97Qs (Myc-mHTT-Q97) and NEs were isolated and the Myc-NT-HTT-Q97 was IP’d with an anti-Myc tag Ab and the Myc-IC was analyzed by WBs to detect FL-PNKP or PNKP domains (lower panel; arrows). (E) BiFC assay of SH-SY5Y cells cotransfected with plasmids: Panel 1) pVN173-PNKP and pVC-BIFC-155, Panel 2) pVN-PNKP and pVC-NT-wtHTT-Q23, Panel 3) pVN-PNKP and pVC-NT-mHTT-Q97, Panel 4) pVN (PHOS + KIN) and pVC-BIFC-155, Panel 5) pVN-(PHOS + KIN) and pVC-NT-wtHTT-Q23, and panel 6) pVN-(PHOS + KIN) and pVC-NT-mHTT-Q97. Reconstitution of fluorescence was monitored via fluorescence microscopy. Nuclei were stained with DAPI (blue).

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Figure 1. Cryo-EM Structures I to V. ; Panels A-E show for each structure: upper row: a schematic of the conformations of the 70S ribosome, tRNA and RF2, with ribosomal subunits (50S and 30S) and A, P and E sites labeled; middle row: structures with RF2 shown in red, tRNAfMet in orange, 30S subunit in yellow, 50S subunit in cyan and mRNA in purple; lower row: cryo-EM density for mRNA, tRNA and RF2, colored as in the middle row. Domains of RF2 are labeled in panel B, illustrating the codon-recognition superdomain (domains 2 and 4) and catalytic domain (3).

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Figure 3. FACT stimulates Ubp10 constructs that lack intrinsic ability to bind nucleosomes. ; (A) Schematic of Ubp10 and its truncations with the locations of the catalytic domain (USP) and an N-terminal region rich in aspartic acid and glutamic acid repeats shown. (B) Native gel showing complexes formed between Ubp10 constructs and ubiquitinated nucleosomes (purified proteins used in this experiment are shown in Figure 3—figure supplement 4). (C) Ubp10 activity was measured as in Figure 2; time points were taken after incubating 2 µM H2A/H2B-Ub dimers with 5 nM Ubp10 fragments. (D) NCP-Ub cleavage activity monitored in the presence and absence of FACT and several Ubp10 constructs.

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Figure 2—figure supplement 2. Cryo-EM density (mesh) shows that the tip of the GGQ loop of RF2 is poorly ordered (gray model) when the catalytic domain adopts the β-hairpin conformation (red; Structure II is shown).

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Figure 2. Rearrangement of the catalytic domain of RF2 upon tRNA displacement from the PTC. ; (A) Rearrangements of RF2 in Structure II (RF2 in red, P-tRNA in orange and 23S in cyan) in comparison with the canonical 70S•RF2 conformation (X-ray structure: Korostelev et al., 2008; RF2 in pink, P-tRNA in light orange, 23S in gray). Arrows show rearrangements for RF2 (red), tRNA acceptor arm and 23S nucleotide A2602 (black). The superposition was achieved by structural alignment of 23S rRNA. (B) The GGQ region of RF2 forms a long β-hairpin that reaches into the constriction of the peptide tunnel (at A2062) and thus plugs the tunnel. Nucleotides of 23S rRNA and the GGQ motif are labeled. (C) Cryo-EM density for the β-hairpin formed by the GGQ region. Residues of RF2 and 23S rRNA (also shown in panel B for reference) are labeled.

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