Journal: bioRxiv

Article Title: PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

doi: 10.1101/2023.05.05.539524

Figure Lengend Snippet: (A) Graph showing the mitochondrial OCR of C2C12 cells overexpressing pMIG GFP and pMIG RBM3-GFP, basal OCR and OCR after treatment with oligomycin (1 uM), FCCP (3 uM), antimycin and rotenone (1.5 uM), where the x-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. (B) Bar graph measuring the basal respiration, maximum respiration (OCR after FCCP addition), spare respiratory capacity (basal respiration-maximum respiration) and ATP-linked respiration (basal respiration-respiration after oligomycin addition) of C2C12 cells overexpressing pMIG-GFP and pMIG-RBM3 GFP where the y-axis represents oxygen consumption rate in pMol/Min (n=2). (C) Heat map showing levels of TCA metabolites using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (D) Heat map showing levels of TCA metabolites using media (48 hrs.) from C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (E) Graphical representation of levels of acetyl-CoA using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). mRNA expression levels of glycolytic genes (F) PKM1 , (G) PKM2 in C2C12 overexpressing pMIG-GFP control and pMIG-RBM3. (H) Western blot analysis of glycolytic protein levels (PKM1, PKM2) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (I) mRNA expression levels of glycolytic genes PKM1 , PKM2 in mouse primary myoblasts overexpressing pMIG-GFP control and pMIG-RBM3 (n=3). (J) Western blot analysis of AMPK-beta and 4E-BP1 using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (K) Western blot analysis of acetyl-CoA carboxylase (ACC) using C2C12 cells overexpressing pMIG-GFP control and pMIG-RBM3. (L) Bar graph quantifying phosphorylated/total 4E-BP1, ACC and AMPK-beta respectively. *, **, *** represents p-value < 0.05, 0.01 and 0.001 respectively.

Article Snippet: Primary antibodies MyHC (Invitrogen 14650382), MF-20 (DHSB AB_2147781) MYOG (Invitrogen MA5-11486), MyoD1 (Santa Cruz SC-377460), RBM3 (Invitrogen PA5-51976), beta-ACTIN (CST 4967S), beta-Tubulin (CST 2146), PKM1(CST D30G6), beta-Actin (CST 4967) PKM2 (CST D78A4), PDH (CST 3205), SDHA (CST 5839), phospho-4E-BP1 (CST 2855), total 4E-BP1 (CST 9452), phospho-AMPK-alpha (CST 2535), total-AMPK-alpha (CST 5831), phospho-AMPK-beta (CST 4186), total-AMPK-beta (CST 4150), phospho-ACC (CST 11818) and total ACC (CST 3676) were used in 1:1000 dilution and incubated at 4 0 C overnight.

Techniques: Control, Expressing, Western Blot

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: Primary mouse hepatocytes were incubated for 1 h with DMSO as vehicle, 10 μM 991, 500 μM phenformin, 2 mM AICAR, 100 μM A769662, 10 nM glucagon or with concentrations of 991 and glucagon as indicated for measurements of intracellular adenine nucleotide concentrations ( a ), AMPK activity by immunoprecipitation using anti-AMPKα1 and anti-AMPKα2 antibodies ( b , d ) and total PDE activity ( d , e ). In e , the 100% value for PDE activity in the DMSO-treated control condition was 6.4±0.7 μU per mg of protein. In c , mouse hepatocytes were incubated for 20 min with 10 μM 991 or DMSO as vehicle before incubation with 10 nM glucagon for ELISA measurements of cAMP concentrations at the indicated times. In f , mouse hepatocytes were incubated for 20 min with 10 μM 991 or DMSO as vehicle and 5 mM pan-PDE inhibitor IBMX before incubation with the indicated concentrations of glucagon for 15 min and measurement of cAMP (ELISA method). Values are means±s.e.m. for n =3 ( a – d ), n =5 ( e ) or n =4 ( f ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations with DMSO.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Incubation, Activity Assay, Immunoprecipitation, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: Primary hepatocytes from wild-type mice or mice bearing a liver-specific deletion of the two AMPK catalytic subunits (AMPK α 1 −/− α LS−/− ) were treated as described in the legends to and . The cells were collected and lysed for immunoblotting levels of phosphorylated ACC and AMPK versus total proteins along with GAPDH as a loading control ( a ). Extracts were also prepared for the measurement of cAMP concentrations by ELISA ( b ), for PKA assay ( c ) and for PDE assay ( d ). Values are means±s.e.m. for n =3 ( b – d ) separate experiments, and in a representative immunoblots are shown. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations with DMSO or between the indicated conditions.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Protein Kinase A Assay

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: PDE4B was cloned from mouse hepatocyte cDNA. The recombinant protein was overexpressed in E. coli and purified. PDE protein was phosphorylated for 1 h with purified recombinant activated AMPK and/or purified PKA catalytic subunits and [γ- 32 P] ATP, and analysed by SDS–PAGE followed by Coomassie blue staining and phosphorimaging for quantification ( a , c ). In b , PDE was phosphorylated for 1 h with recombinant activated AMPK and [γ- 32 P]. Phosphorylation sites were identified by LC–MS/MS after trypsin digestion and radioactive peak separation by high-performance liquid chromatography (HPLC). The phosphorylation sites that were identified are underlined in the right hand panel. In d and e , recombinant PDE was phosphorylated as above but with non-radioactive ATP for PDE assay as indicated. In d , separate determinations of V max and K M were made by linear regression of double reciprocal (Lineweaver Burk) plots. In e , the basal PDE activities of the wild-type (WT), S118A, S125A and S304A mutant proteins were 1.97±0.25, 0.14±0.01, 1.59±0.15 and 0.32±0.09 mU per mg of protein, respectively. Values are means±s.e.m. for n =3 ( c – e ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05) compared with control incubations or between the indicated conditions.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Clone Assay, Recombinant, Purification, SDS Page, Staining, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, High Performance Liquid Chromatography, Mutagenesis, Control

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: In a , wild-type (WT) or mutant recombinant mouse liver PDE4B was incubated for 1 h with non-radioactive ATP in the presence (+) or absence (−) of recombinant activated AMPK. Proteins (0.1 μg) were seperated by SDS–PAGE for immunoblotting with the indicated antibodies. In b and c , mouse hepatocytes from either WT ( b ) or both WT and AMPK α 1 −/− α 2 LS−/− mice ( c ) were serum-starved overnight and incubated for 1 h with the indicated concentrations of 991 or phenformin. The cells were collected and lysed for immunoblotting with the indicated antibodies, except for PDE4B, which was immunoprecipitated as described in the Methods section, before immunoblotting. In c , phosphorylation levels of AMPK and its targets ACC, Raptor and PDE4B were quantified by densitometry and expressed relative to the corresponding total protein levels or GAPDH before normalization as indicated. Representative immunoblots are shown and for blot quantification in c , the values are means±s.e.m. for n =3 (p-ACC, p-Raptor and p-AMPK) or n =4 (p-PDE4B) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P <0.05).

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Mutagenesis, Recombinant, Incubation, SDS Page, Western Blot, Immunoprecipitation, Phospho-proteomics

Journal: Nature Communications

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

doi: 10.1038/ncomms10856

Figure Lengend Snippet: Unlike biguanides, treatment with 991 activates AMPK without increasing cellular AMP levels. Both biguanides and 991 activate the major PDE 4B isoenzyme in hepatocytes in an AMPK-dependent manner. Metformin and phenformin activate hepatic AMPK either directly or via a rise in AMP, which could compete with ATP to inhibit adenylate cyclase. Phosphorylation-induced activation of PDE4B by AMPK reduces glucagon-stimulated cAMP accumulation. As a consequence, PKA activation by glucagon and downstream signalling are decreased in hepatocytes incubated with 991, the effect being AMPK-dependent.

Article Snippet: Anti-total ACC (Merck Millipore, Catalogue No. 04-322), anti-P-Ser79-ACC (Merck-Millipore, Catalogue No. 07-303), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Merck-Millipore, Catalogue No. MAB374), anti-total GP (Sigma, Catalogue No. HPA000962), anti-total AMPK β1(R&D Systems, Catalogue No. AF2854) and anti-total AMPK β2 (R&D Systems, Catalogue No. MAB3808), anti-PThr172-AMPK (T172) (Cell Signaling Technologies, Catalogue No. 2535, anti-P-AMPK-substrate (Cell Signaling Technologies, Catalogue No. 5759), anti-P-PKA-substrate (Cell Signaling Technologies, Catalogue No. 9624), anti-total Raptor (Cell Signaling Technologies, Catalogue No. 2280) and anti-P-Ser792-Raptor (Cell Signaling Technologies, Catalogue No. 2083), anti-total CREB (Cell Signaling Technologies, Catalogue No. 9197), anti-phospho-Ser133-CREB (Cell Signaling Technologies, Catalogue No. 9198) and anti-total PDE4B (Origene, Catalogue No. TA503471) antibodies were from the sources cited.

Techniques: Phospho-proteomics, Activation Assay, Incubation

Journal: Science Advances

Article Title: Conformational changes in the yeast mitochondrial ABC transporter Atm1 during the transport cycle

doi: 10.1126/sciadv.abk2392

Figure Lengend Snippet: ( A ) Cryo-EM structures of Atm1 in the absence (left) and presence of AMP-PNP-Mg 2+ (right, MSP1E3D1 structure) in the same colors as in except for the C-terminal helices (orange and coral; viewed from the matrix). Blue and green arrows denote the movement of the NBDs toward each other, and gray arrows indicate a possible path for the helix rearrangement. ( B ) At the C terminus of AMP-PNP-Mg 2+ –bound Atm1, Q674, E675, and D676 and the side chains of R634 (shown as sticks) form intra- and interchain hydrogen and ionic bonds (dotted brown lines). Side chains of A637 and L657 and the segments around them (purple) would clash if the C-terminal helices were to move as rigid bodies. ( C ) Detail of (B). ( D ) Negative-stain EM 2D class averages show the Atm1-L657 truncation mutant reconstituted into MSP1D1 nanodiscs with or without added AMP-PNP and MgCl 2 .

Article Snippet: Cryo-EM datasets were recorded on an FEI Krios G3i microscope equipped with a Gatan K3 direct electron detector (yeast Atm1 nucleotide-free state: 0.837 Å per pixel, 25-eV energy filter slit width, 64 electrons/Å 2 total dose, 3-s exposure time, 50 frames per movie, 2430 + 1690 micrographs; CtAtm1: 0.837 Å per pixel, 30-eV energy filter slit width, 63 electrons/Å 2 total dose, 3-s exposure time, 50 frames per movie, 2066 images) or an FEI Falcon 3EC camera (AMP-PNP–bound state, MSP1D1; 0.833 Å per pixel, 50 electrons/Å 2 total dose, 41-s exposure time, 53 frames per movie, 4610 + 904 micrographs), or on an FEI Krios G2 microscope with a Gatan K2 direct electron detector (AMP-PNP–bound state, MSP1E3D1; 0.828 Å per pixel, 30-eV energy filter slit width, 68 electrons/Å 2 total dose, 8-s exposure time, 40 frames per movie, 1885 micrographs), all using the automated data acquisition software EPU (Thermo Fisher Scientific).

Techniques: Cryo-EM Sample Prep, Staining, Mutagenesis