Proteomic and Metabolic Analyses of S49 Lymphoma Cells Reveal Novel Regulation of Mitochondria by cAMP and Protein Kinase A

Wilderman, Andrea; Guo, Yurong; Divakaruni, Ajit S.; Perkins, Guy; Zhang, Lingzhi; Murphy, Anne N.; Taylor, Susan S.; Insel, Paul A.

doi:10.1074/jbc.m115.658153

Cited by 9 publications

(7 citation statements)

References 51 publications

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“…6h). Fatty acids taken up by the cell, synthesized, or derived from lipolysis can be broken down through FAO, a process in which PKA has also been implicated 42–44 . Correspondingly, assessment of CO 2 release from 14 C-labelled oleate in KGC cultures ± Dox demonstrated that GNAS R201C was required to sustain FAO activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism

et al. 2018

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G protein α (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that Gnas cooperates with Kras to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1-3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.

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Section: Resultsmentioning

confidence: 99%

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism

et al. 2018

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“…The technique involves selective permeabilization of the plasma membrane to release soluble enzymes from the cytoplasm, for which we use rPFO as in previous work [2,4,7,10–12]. rPFO is a recombinant, mutant form of perfringilysin O, a cholesterol-dependent cytolysin.…”

Section: Discussionmentioning

confidence: 99%

In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents

Divakaruni

Andreyev

Rogers

et al. 2018

Analytical Biochemistry

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Activities of enzymes localized to the mitochondrial matrix of mammalian cells are often critical regulatory steps in cellular metabolism. As such, measurement of matrix enzyme activities in response to genetic modifications or drug interventions is often desired. However, measurements in intact cells are often hampered by the presence of other isozymes in the cytoplasm as well as the inability to deliver enzyme substrates across cellular membranes. Classic approaches to liberate matrix enzymes utilize harsh treatments that disrupt intracellular architecture or require significant starting material to allow mitochondrial isolation prior to sample extraction. We describe a method using permeabilization reagents for both the plasma and mitochondrial membranes to allow in situ measurement of matrix enzyme activities. It is applied to adherent cell monolayers in 96-well plates treated with perfringolysin O to permeabilize the plasma membrane and alamethicin to permeabilize the mitochondrial inner membrane. We present three examples validated with inhibitor sensitivity: (i) Complex I-mediated oxygen consumption driven by NADH, (ii) ATP hydrolysis by the FF complex measuring pH changes in an Agilent Seahorse XF Analyzer, and (iii) Mitochondrial glutaminase (GLS1) activity in a coupled reaction monitoring NADH fluorescence in a plate reader.

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“…A number of mitosome-associated proteins are closely related to those of mitochondria [ 73 ]. Our tentative association of GlPDE with mitosomes may represent a further similarity with mitochondria of higher eukaryotes, where cAMP synthesis and hydrolysis is an important regulatory circuit for mitochondrial metabolism [ 74 , 75 ]. Since mitosomes do not occur in mammalian cells, but are essential for Giardia and presumably regulated by cAMP signaling, future studies to verify the localization of GlPDE are certainly warranted.…”

Section: Discussionmentioning

confidence: 99%

The single cyclic nucleotide-specific phosphodiesterase of the intestinal parasite Giardia lamblia represents a potential drug target

et al. 2017

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BackgroundGiardiasis is an intestinal infection correlated with poverty and poor drinking water quality, and treatment options are limited. According to the Center for Disease Control and Prevention, Giardia infections afflict nearly 33% of people in developing countries, and 2% of the adult population in the developed world. This study describes the single cyclic nucleotide-specific phosphodiesterase (PDE) of G. lamblia and assesses PDE inhibitors as a new generation of anti-giardial drugs.MethodsAn extensive search of the Giardia genome database identified a single gene coding for a class I PDE, GlPDE. The predicted protein sequence was analyzed in-silico to characterize its domain structure and catalytic domain. Enzymatic activity of GlPDE was established by complementation of a PDE-deficient Saccharomyces cerevisiae strain, and enzyme kinetics were characterized in soluble yeast lysates. The potency of known PDE inhibitors was tested against the activity of recombinant GlPDE expressed in yeast and against proliferating Giardia trophozoites. Finally, the localization of epitope-tagged and ectopically expressed GlPDE in Giardia cells was investigated.ResultsGiardia encodes a class I PDE. Catalytically important residues are fully conserved between GlPDE and human PDEs, but sequence differences between their catalytic domains suggest that designing Giardia-specific inhibitors is feasible. Recombinant GlPDE hydrolyzes cAMP with a Km of 408 μM, and cGMP is not accepted as a substrate. A number of drugs exhibit a high degree of correlation between their potency against the recombinant enzyme and their inhibition of trophozoite proliferation in culture. Epitope-tagged GlPDE localizes as dots in a pattern reminiscent of mitosomes and to the perinuclear region in Giardia.ConclusionsOur data strongly suggest that inhibition of G. lamblia PDE activity leads to a profound inhibition of parasite proliferation and that GlPDE is a promising target for developing novel anti-giardial drugs.

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Proteomic and Metabolic Analyses of S49 Lymphoma Cells Reveal Novel Regulation of Mitochondria by cAMP and Protein Kinase A

Cited by 9 publications

References 51 publications

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism

In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents

The single cyclic nucleotide-specific phosphodiesterase of the intestinal parasite Giardia lamblia represents a potential drug target

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