Journal: Molecular Metabolism

Article Title: Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

doi: 10.1016/j.molmet.2024.102018

Figure Lengend Snippet: G-1,6-BP regulates mitochondrial pyruvate uptake. ( A ) Protein from mouse brain mitochondria was isolated and probed for mitochondrial markers (ATPB and Cox IV) and PGM2L1, which was localized to both, the cytosolic (Cyto) and mitochondrial fraction (Mito). Blot is representative of 3 separate experiments. ( B ) The mitochondrial G-1,6-BP content remained stable over a 15 min incubation period (unpaired t-test, P > 0.05). ( C ) Addition of G-1,6-BP (100 μM) to pyruvate dehydrogenase (PDH) did not alter its activity (paired t-test, P > 0.05). ( D ) A DARTS assay was performed with mouse hippocampal protein extracts incubated with low (-PI) or high protease inhibitor cocktail (+PI) after addition of vehicle (double distilled H 2 O, 0 mM) or 0.5 mM or 1 mM G-1,6-BP. G-1,6-BP (0.5 mM and 1 mM) protected ( E ) MPC2 from degradation by endogenous proteases (one way ANOVA with Holm-Šídák's multiple comparisons test: (∗∗) P < 0.01), while ( F ) MPC1 was only protected by 1 mM G-1,6-BP (one way ANOVA with Holm-Šídák's multiple comparisons test: (∗∗∗) P < 0.001, (ns) not significant). The protective effects were lost upon incubation with high PI (+PI) cocktail. ( G ) Control plasmids (empty vector, Co Plasmid) or human ( h ) PGM2L1 were overexpressed in human HEK cells expressing MPC1-mVenus (V)/MPC2-RLuc8 (R). Blot is representative of 3 separate experiments. ( H ) Transfected HEK MPC1V/MPC2R were treated with pyruvate (5 mM, arrowhead) or PBS and the BRET ratio was monitored. Values are expressed as fold of baseline with the PBS values subtracted (see Supplemental Figs. 3E and F for raw values). Areas marked in grey were used for analysis (N = 3). ( I ) The mean of 2 values (baseline and 15 min after addition of pyruvate) and the first value after addition of pyruvate of the areas marked in grey in H were compared (two way ANOVA Plasmid (∗∗∗∗) P < 0.0001, treatment (∗∗∗) P < 0.001, interaction P > 0.05, Holm-Šídák's multiple comparisons test (∗) P < 0.05). ( J ) Brain mitochondria were isolated and 14 C 3 -pyruvate uptake was measured in the presence of vehicle (double distilled H 2 O, control, N = 8), 100 μM G-1,6-BP (N = 6) or 10 μM UK-5099 (N = 4). All values were related to each respective time point 0 (0 min, which was immediately stopped by the addition of 10 μM UK-5099). Pyruvate uptake with 0 and 100 μM G-1,6-BP was compared (two way ANOVA, Time (∗∗∗) P < 0.001, Treatment (∗∗) P < 0.01, Interaction P > 0.05, Holm-Šídák's multiple comparisons test: (∗) P < 0.5). ( K ) Mouse brain mitochondria were isolated and analyzed for their oxygen consumption rates (OCR). Mitochondria were stimulated with pyruvate (5 mM), ADP (4 mM) and malate (1 mM) and the effect of 100 μM G-1,6-BP on pyruvate-mediated OCR was analyzed. Raw relative fluorescence units (RFU) values are shown. ( L ) The slopes of each curve (from 1 to 45 min, shown in K) of the response was analyzed (unpaired t-test, (∗) P < 0.05). All data are presented as scatter blot with mean ± S.E.M., except for H, J and K were only mean ± S.E.M is shown.

Article Snippet: Human ( h ) PKN1 was purchased from GeneCopoeia (Rockville, MD, USA) and subcloned into the mammalian expression vector pTO_HA_StrepIII_c_GW_FRT_EF1alpha as described in [ ].

Techniques: Isolation, Incubation, Activity Assay, Protease Inhibitor, Control, Plasmid Preparation, Expressing, Transfection, Fluorescence

Journal: Molecular Metabolism

Article Title: Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

doi: 10.1016/j.molmet.2024.102018

Figure Lengend Snippet: Regulation of PGM2L1 activity. ( A ) Human PGM2L1 protein structure was predicted with AlphaFold Monomer v2.0 pipeline. The amino acids T173-H176 are shown in green in the predicted structure and encompass the predicted phosphorylation site S175. ( B ) Human SH-SY5Y cells were transfected with empty vectors (EV for the PKN1 and PGM2L1 plasmids; Control), HA-tagged PKN1+EV (for PGM2L1 plasmid), untagged PGM2L1+EV (for PKN1 plasmid) and PKN1+PGM2L1 and G-1,6-BP levels were analyzed (one way ANOVA with Holm-Šídák's multiple comparisons test ((∗) P < 0.05, (∗∗∗) P < 0.001). Blot is representative of 3 separate experiments. ( C ) The top 50 (rank) kinases predicted to phosphorylate human PGM2L1 S175 were analyzed with WebGestalt, using Network-Topology based analysis and PPI Biogrid. A pie chart summarizing the enrichment ratio (% of total) of the top 10 predicted networks are shown. ( D ) Scheme summarizing the metabolic processes involved in PGM2L1 regulation. Glycolysis, shown in green, has a stimulatory- and metabolic stress an inhibitory effect on G-1,6-BP levels, due to degradation by PMM1. Additionally PGM2L1 is inhibited by citrate, encompassing a potential feedback inhibition. The phosphorylation of S175 is further predicted to inhibit PGM2L1 activity. All data are presented as scatter blot with mean ± S.E.M. A and D were prepared in BioRender.

Article Snippet: Human ( h ) PKN1 was purchased from GeneCopoeia (Rockville, MD, USA) and subcloned into the mammalian expression vector pTO_HA_StrepIII_c_GW_FRT_EF1alpha as described in [ ].

Techniques: Activity Assay, Transfection, Control, Plasmid Preparation, Inhibition

Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Figure Lengend Snippet: The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and -3xrGBD, and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.

Article Snippet: The following plasmids, generated in this study, are available on Addgene ( http://www.addgene.org/ ): 129625 , dTomato-2xrGBD (C1 vector); 176098 , dimericTomato-2xrGBD (pLV vector); 129624 , mNeongreen-2xrGBD; 176091 , mNeonGreen-3xrGBD; 129633 , mNeonGreen-aGBD (anillin); 129634 , mNeonGreen-pGBD (PKN1 codon optimized); 176094 , H2A-mTurquoise2-CDC42-G12V-ΔCaaX; 176095 , H2A-mTurquoise2-RAC1-G12V-ΔCaaX; and 176097 , H2A-mTurquoise2-RHOA-G14V-ΔCaaX.

Techniques: Expressing, Transfection, Protein Binding

Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Figure Lengend Snippet: The GBDs of anillin and PKN1 are not suitable for a relocation Rho sensor. (A) Change in cytosolic intensity for CMVdel-mNeonGreen-1xpGBD, -2xpGBD and -3xpGBD, and CMVdel-dimericTomato-2xpGBD co expressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of samples per condition is: dT-2xpGBD=16, mNG-1xpGBD=24, mNG-2xpGBD=28, mNG-3xpGBD=14. (B) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-2xpGBD in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells from a single replicate per condition is: H2A-dT-2xpGBD=19, H2A-RhoA-dT2xpGBD=16. (C) Change in cytosolic intensity for CMVdel-mNeonGreen-1xaGBD, -2xaGBD and -3xaGBD, CMVdel-dimericTomato-1xaGBD and eGFP-anillin(AHD+PH) coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells per condition is: dT-1xaGBD=14, eGFP-AHD+PH=27, mNG-1xaGBD=17, mNG-2xaGBD=10, mNG-3xaGBD=13. (D) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-1xaGBD and mScarlet-I-anillin(AHD+PH) in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX-expressing HeLa cells. The dashed line indicates a ratio of one. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells from a single replicate per condition is: H2A-aGBD=12, H2A-RhoA-aGBD=10, H2A-RhoA-AHD+PH=13. (E) Amino acid sequence alignment for aGBD, rGBD and pGBD from MUSCLE and depicted with the clustalX color code, where green is polar, blue is hydrophobic, purple is negative charge, red is positive charge, yellow is prolines, orange is glycines, cyan is aromatic and white is unconserved. (F) Crystal structures of PKN1 G-protein binding domain (purple) and anillin G-protein binding domain (yellow), bound to RhoA GTP (gray). On the left, a structural alignment of PKN1 and anillin by their G-protein binding domains. On the right, a structural alignment of the bound RhoA molecules, showing the two binding positions at the RhoA molecule (PDB: anillin, 4XOI ; PKN1, 1CXZ ).

Article Snippet: The following plasmids, generated in this study, are available on Addgene ( http://www.addgene.org/ ): 129625 , dTomato-2xrGBD (C1 vector); 176098 , dimericTomato-2xrGBD (pLV vector); 129624 , mNeongreen-2xrGBD; 176091 , mNeonGreen-3xrGBD; 129633 , mNeonGreen-aGBD (anillin); 129634 , mNeonGreen-pGBD (PKN1 codon optimized); 176094 , H2A-mTurquoise2-CDC42-G12V-ΔCaaX; 176095 , H2A-mTurquoise2-RAC1-G12V-ΔCaaX; and 176097 , H2A-mTurquoise2-RHOA-G14V-ΔCaaX.

Techniques: Binding Assay, Expressing, Sequencing, Protein Binding

Journal: Journal of Clinical Pathology

Article Title: Microbial infections in eight genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis

doi: 10.1136/jcp.2009.072561

Figure Lengend Snippet: CFS/ME-associated genes and transcription factors in patients with CFS/ME, Q-fever-associated CFS/ME and endogenous depression

Article Snippet: PKN1 , NM_213560 , Hs00177028_m1 , 4.58 , 0.0003 , 3.95 , 0.01 , 1.03 , 0.887.

Techniques:

Journal: Journal of Clinical Pathology

Article Title: Microbial infections in eight genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis

doi: 10.1136/jcp.2009.072561

Figure Lengend Snippet: Fold-difference values for 88 genes in each of eight subtypes (A–H) in 114 subtyped patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME). Genes without values for the subtypes are those for which there was missing data for one or more subtypes. Bold type indicates genes targeted by existing drugs and those CFS/ME subtypes in which fold-difference values of 1.5 were found

Article Snippet: PKN1 , NM_213560 , Hs00177028_m1 , 4.58 , 0.0003 , 3.95 , 0.01 , 1.03 , 0.887.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

doi: 10.15252/emmm.201910547

Figure Lengend Snippet: A PKN1 KO clones expressing p.M694V or WT Pyrin were obtained and confirmed by Western blot analysis. Four clones are shown. Clones 3 and 6 were selected. B Absolute transcript level of the indicated genes was quantified by RNAseq in U937 cells (Benaoudia et al , ) and expressed as count per million (cpm). PYCARD encodes ASC and is shown as a reference. C PKN2 transcript levels were assessed in U937 cells at 24 h post‐electroporation with the indicated non‐targeting (NT) siRNA or three different siRNAs targeting PKN2 . D–F U937 cells with the indicated Pyrin variant were treated (+Dox, plain lines) or not (No Dox, dotted lines) with doxycycline (Dox) at 24 h post‐electroporation with the indicated siRNA. Cell death was monitored every 15 min. (F) SiRNA 14 did not reduce substantially PKN2 levels. Data information: (D, E, F) Cell death was normalized to the maximal propidium iodide (PI) incorporation determined using TX‐100‐treated cells. Each dot represents the mean ± SD of a biological triplicate from one experiment representative of three independent experiments. Source data are available online for this figure.

Article Snippet: The sgRNA targeting PKN1 and PKN2 ( ) were selected from the Brunello library (Addgene) and cloned into the PGKpuro2ABFP vector (from Kosuke Yusa; Addgene plasmid # 50946). sgRNA plasmids were transduced in a previously described Cas9‐expressing U937 clone (Lagrange et al , ) by spinoculation.

Techniques: Clone Assay, Expressing, Western Blot, Electroporation, Variant Assay

Journal: Nature Communications

Article Title: Exome-wide analysis identifies three low-frequency missense variants associated with pancreatic cancer risk in Chinese populations

doi: 10.1038/s41467-018-06136-x

Figure Lengend Snippet: The identified variants associated with pancreatic cancer risk in the discovery, replication and combined samples

Article Snippet: The siRNA oligonucleotides targeting PKN1 and non-targeting siRNA (Supplementary Table ) were purchased from RiboBio (Guangzhou, China).

Techniques:

Journal: Nature Communications

Article Title: Exome-wide analysis identifies three low-frequency missense variants associated with pancreatic cancer risk in Chinese populations

doi: 10.1038/s41467-018-06136-x

Figure Lengend Snippet: PKN1 rs34309238 variant influences pancreatic cancer risk by altering the level of phosphorylated PKN1 and thus affecting the FAK/PI3K/AKT signalling pathway. a Protein modification sites of PKN1. Annotations were obtained from the PhosphoSitePlus database. b Result of the iTRAQ-based comparative proteomics screen. PANC-1 cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector. The raw intensity values of cells transfection with PKN1[A] or PKN1[C] were divided by the intensity values of cells transfection with control vector to obtain the relative intensity values. The y axis shows the relative intensity values of cells' transfection with PKN1[A] minus the relative intensity values of cells' transfection with PKN1[C]. The x axis shows the molecular weight of detected peptides. The proteomics screen experiment was repeated independently for two times with similar results. c Levels of phosphorylated FAK and AKT were affected by the PKN1 rs34309238 variant. Cells were seeded in six-well plates after transfection with PKN1[A], PKN1[C] or control vector (left) and PKN1-targeting siRNAs or control siRNA (right). d Levels of phosphorylated FAK and AKT were reduced by the PKN1 inhibitors. Cells were seeded in six-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For c , d , the western blot experiment was repeated independently for three times with similar results

Article Snippet: The siRNA oligonucleotides targeting PKN1 and non-targeting siRNA (Supplementary Table ) were purchased from RiboBio (Guangzhou, China).

Techniques: Variant Assay, Modification, Multiplex sample analysis, Transfection, Control, Plasmid Preparation, Molecular Weight, Western Blot

Journal: Nature Communications

Article Title: Exome-wide analysis identifies three low-frequency missense variants associated with pancreatic cancer risk in Chinese populations

doi: 10.1038/s41467-018-06136-x

Figure Lengend Snippet: PKN1 rs34309238 variant influences pancreatic cancer cells' proliferation. a , b Overexpression of PKN1[A] substantially enhanced the rate of cell proliferation in PANC-1 ( a ) and BxPC-3 ( b ) cells. Cells were seeded in 96-well plates after transfection with PKN1[A], PKN1[C] or control vector. c , d Knockdown of PKN1 significantly reduced the proliferation of PANC-1 ( c ) and BxPC-3 ( d ) cells. Cells were seeded in 96-well plates after transfection with PKN1-targeting siRNAs or control siRNA (siControl). e , f PKN1 inhibitors significantly reduced the rate of cell proliferation in PANC-1 ( e ) and BxPC-3 ( f ) cells. Cells were seeded in 96-well plates after transfection with PKN1 inhibitors Lestaurtinib and Ro318220 or DMSO as control. For a – f , cell numbers were determined every 24 h for 96 h using CCK-8 assays and the results present means ± s.e.m. from three independent experiments and each had six replications. * P < 0.05, ** P < 0.01, compared with the control by two-sided unpaired Student’s t test

Article Snippet: The siRNA oligonucleotides targeting PKN1 and non-targeting siRNA (Supplementary Table ) were purchased from RiboBio (Guangzhou, China).

Techniques: Variant Assay, Over Expression, Transfection, Control, Plasmid Preparation, Knockdown, CCK-8 Assay