Journal: Cells

Article Title: Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA

doi: 10.3390/cells10123354

Figure Lengend Snippet: Mitochondrial proteins (UniProt-IDs) with significant downregulation (upper part) and upregulation (lower part) in ClpP-null MEFs versus WT (3 versus 3). Factors with special relevance for this manuscript are highlighted in bold letters. Previously reported candidate ClpP cleavage substrates in mouse are identified at the right margin with citations of the relevant PubMed-ID number.

Article Snippet: The following Taqman assays (ThermoFisher, Waltham, MA, USA) were employed for murine transcripts: ClpP : Mm00489940_m1, ClpX : Mm00488586_m1, Poldip2 : Mm00458936_m1, Lrpprc : Mm00511512_m1, Grsf1 : Mm00618579_m1, Gfm1 : Mm00506856_m1, Chchd2 : Mm01742631_s1, Mut : Mm00485312_m1, Oat : Mm00497544_m1, Tbp : Mm00446973_m1.

Techniques:

Journal: Cells

Article Title: Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA

doi: 10.3390/cells10123354

Figure Lengend Snippet: Schematic overview of potential mechanisms in the presence ( A ) and absence ( B ) of ClpP. ClpP inactivating variants act via ClpX accumulation on mitochondrial nucleoid/RNA granule components, triggering excess POLDIP2, LRPPRC, GFM1, GRSF1, and mtDNA abundance, as well as an enlarged nucleoid area, as a clear result of this study. Less consistent across cell types and potentially without ClpX interactions, multimerizing enzymes in the matrix also show increased abundance. In parallel, elevated levels of varying molecular chaperones suggest a mitochondrial unfolded protein response and aggregation tendencies. OAT multimer and nucleoid shapes were adapted from Refs. [ , ].

Article Snippet: The following Taqman assays (ThermoFisher, Waltham, MA, USA) were employed for murine transcripts: ClpP : Mm00489940_m1, ClpX : Mm00488586_m1, Poldip2 : Mm00458936_m1, Lrpprc : Mm00511512_m1, Grsf1 : Mm00618579_m1, Gfm1 : Mm00506856_m1, Chchd2 : Mm01742631_s1, Mut : Mm00485312_m1, Oat : Mm00497544_m1, Tbp : Mm00446973_m1.

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transcriptomics based identification of S100A3 as the key anti-hepatitis B virus factor of 16F16.

doi: 10.1016/j.biopha.2023.114904

Figure Lengend Snippet: Fig. 8. qPCR validation of DEGs. (A) qPCR analysis of upregulated genes selected from transcriptomics analysis. qPCR analysis results were consistent with the transcriptomics analysis. (B) Validation of downregulated genes through qPCR analysis, results were consistent with the transcriptomics analysis which shows ac curacy of transcriptomics data. (C) Western blot validation of EREG, PDIA2 and PDIA6.

Article Snippet: The membranes were incubated with the primary S100A3 antibody (sc-514339, 1:1000), PDIA2 antibody (BST19364904, BOSTER Biological Technology), PDIA6 antibody (A03813–2, BOSTER Biological Technology), EREG antibody (ZP3681BP81, BOSTER Biological Technology) and GAPDH (GB12002, 1/1000) overnight at 4◦C.

Techniques: Biomarker Discovery, Western Blot

Journal: bioRxiv

Article Title: Leigh Syndrome-inducing Mutations Affect LRPPRC / SLIRP Complex Formation

doi: 10.1101/2020.04.16.044412

Figure Lengend Snippet: Identification of the minimal region of LRPPRC required to form a stable interaction with SLIRP. A) Schematic view of the LRPPRC proteins with domain annotations. Using secondary structure prediction server, LRPPRC was divided in several fragments. Three domains are formed of PPR motifs while three inter-domains present helical repeats of a different type. Each domain is colored differently. We generated a series of constructs encompassing one or several of these annotated regions. On the right of the diagrams, a table summarizes the complex formation observed between each LRPPRC construct and the GST-tagged SLIRP protein (Y for yes and N for no). B) Affinity purification of the LRPPRC constructs which were co-expressed with a GST-tag version of its partner SLIRP. GST pull-down were performed as described in the material and method section and demonstrated that the N-terminal fragment NT2 is sufficient to observe a stable complex. Input and bound fractions are shown. HL FL stands for His-tag LRPPRC full length; HL NT1 for His-tag N-terminal fragment 1; HL NT2: His-tag N-terminal fragment 2; HL NCD: His-tag N-terminal and central domain; HL CD: His-tag central domain; HL CT1: His-tag C-terminal fragment CT1; HL CT2: His-tag C-terminal fragment 2 and GS: GST-tag SLIRP.

Article Snippet: The human cDNA clones of LRPPRC and SLIRP were provided by OriGene (product numbers: SC120536 and SC108657).

Techniques: Generated, Construct, Affinity Purification

Journal: bioRxiv

Article Title: Leigh Syndrome-inducing Mutations Affect LRPPRC / SLIRP Complex Formation

doi: 10.1101/2020.04.16.044412

Figure Lengend Snippet: LRPPRC/SLIRP complex stability is almost independent of salt concentration. Cells were lysed in identical buffer with the exception to their respective salt concentrations. Washes were also performed with the same salt concentrations. The bound proteins were finally eluted with 20 mM of reduced glutathione and loaded onto a 12% SDS-PAGE. HL NT1 and GS are as in .

Article Snippet: The human cDNA clones of LRPPRC and SLIRP were provided by OriGene (product numbers: SC120536 and SC108657).

Techniques: Concentration Assay, SDS Page

Journal: bioRxiv

Article Title: Leigh Syndrome-inducing Mutations Affect LRPPRC / SLIRP Complex Formation

doi: 10.1101/2020.04.16.044412

Figure Lengend Snippet: Effect of the LSFC-inducing A354V mutation on LRPPRC/SLIRP complex formation. A) The NT1 construct containing the A354V mutation was co-expressed with the GST-SLIRP protein and pull-down performed. While GST-SLIRP was binding the GST column, the upper band corresponding to the NT1 A354V construct was hardly retained, independently of the salt concentration, indicating that mutated LRPPRC protein does not bind stably to SLIRP anymore. HL NT1 A354V mut stands for His-tag N-terminal fragment 1 carrying the A354V substitution and GS: GST-tag SLIRP protein. B) LRPPRC NT1 structure prediction. The NT1 fragment sequence (residues 60-498) was modeled by the webserver Phyre. The best structural prediction is shown here and was built onto the atomic structure of the PPR10 protein from maize (PDB ID 4M57). The helices surrounding the A354V mutation are shown in the inset. The Alanine residue 354 is shown as stick and colored in orange.

Article Snippet: The human cDNA clones of LRPPRC and SLIRP were provided by OriGene (product numbers: SC120536 and SC108657).

Techniques: Mutagenesis, Construct, Binding Assay, Concentration Assay, Stable Transfection, Sequencing

Journal: bioRxiv

Article Title: Leigh Syndrome-inducing Mutations Affect LRPPRC / SLIRP Complex Formation

doi: 10.1101/2020.04.16.044412

Figure Lengend Snippet: Effect of minor Leigh Syndrome mutations on LRPPRC/SLIRP complex formation and protein stability. A) Pull-down experiments between the full-length LRPPRC constructs containing the different deletions and the GST-SLIRP protein. The constructs with delV866 and delK909 do not impair the LRPPRC/SLIRP complex formation while the construct with delR1276_K1300 appears degraded although it is still pull-downed by GST-SLIRP. WT, delV866, delK909 and delR1276_K1300 stand for wild type LRPPRC, LRPPRC deleted of residue V866, of residue K909 and of the fragment between residues R1276 and K1300 respectively. GS is as . B) and C) Structure prediction of second and third PPR fragments of LRPPRC, residues 504 to 948 and 915 to 1394 respectively. The CD and the CT domain sequences were modeled as in . The mutated amino acids V866 and K909 or the deletion from R1276 to K1300 are highlighted in orange.

Article Snippet: The human cDNA clones of LRPPRC and SLIRP were provided by OriGene (product numbers: SC120536 and SC108657).

Techniques: Construct