- Long-term outcomes in a 25-year-old female affected with lipin-1 deficiency. [Case Reports]
- JRJIMD Rep 2019; 46(1):4-10
- Lipin-1 is a phosphatidic acid phosphohydrolase (EC 18.104.22.168) that catalyzes the dephosphorylation of phosphatidic acid to diacylglycerol and inorganic phosphate. Deficiency of this enzyme causes pote…
Lipin-1 is a phosphatidic acid phosphohydrolase (EC 22.214.171.124) that catalyzes the dephosphorylation of phosphatidic acid to diacylglycerol and inorganic phosphate. Deficiency of this enzyme causes potentially fatal severe, recurrent episodes of rhabdomyolysis triggered by infection. The defect has only recently been recognized so little is known about the long-term outcome in adult patients with this disorder. We report the course and outcome of a 25-year-old female patient with lipin-1 deficiency after a recent episode of rhabdomyolysis requiring intensive care admission with a peak creatine kinase of 500 000 IU/L. One-year post discharge from intensive care, the patient has residual drop foot bilaterally consistent with bilateral common peroneal neuropathies in addition to a background residual distal myopathy.
- Hepatocyte PRMT1 protects from alcohol induced liver injury by modulating oxidative stress responses. [Journal Article]
- SRSci Rep 2019 Jun 24; 9(1):9111
- Protein Arginine methyltransferase 1 (PRMT1) is the main enzyme of cellular arginine methylation. Previously we found that PRMT1 activity in the liver is altered after alcohol exposure resulting in e…
Protein Arginine methyltransferase 1 (PRMT1) is the main enzyme of cellular arginine methylation. Previously we found that PRMT1 activity in the liver is altered after alcohol exposure resulting in epigenetic changes. To determine the impact of these PRMT1 changes on the liver's response to alcohol, we induced a hepatocyte specific PRMT1 knockout using AAV mediated Cre delivery in mice fed either alcohol or control Lieber-DeCarli liquid diet. We found that in alcohol fed mice, PRMT1 prevents oxidative stress and promotes hepatocyte survival. PRMT1 knockout in alcohol fed mice resulted in a dramatic increase in hepatocyte death, inflammation and fibrosis. Additionally, we found that alcohol promotes PRMT1 dephosphorylation at S297. Phosphorylation at this site is necessary for PRMT1-dependent protein arginine methylation. PRMT1 S297A, a dephosphorylation mimic of PRMT1 had reduced ability to promote gene expression of pro-inflammatory cytokines, pro-apoptotic genes BIM and TRAIL and expression of a suppressor of hepatocyte proliferation, Hnf4α. On the other hand, several functions of PRMT1 were phosphorylation-independent, including expression of oxidative stress response genes, Sod1, Sod2 and others. In vitro, both wild type and S297A PRMT1 protected hepatocytes from oxidative stress induced apoptosis, however S297D phosphorylation mimic PRMT1 promoted cell death. Taken together these data suggest that PRMT1 is an essential factor of liver adaptation to alcohol; alcohol-induced dephosphorylation shifts PRMT1 toward a less pro-inflammatory, more pro-proliferative and pro-survival form.
- Characterization of metal binding of bifunctional kinase/phosphatase AceK and implication in activity modulation. [Journal Article]
- SRSci Rep 2019 Jun 24; 9(1):9198
- A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability…
A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability. Herein we report the crystal structure of AceK in complex with ADP and Mn2+ ions. Although the overall structure is similar to the previously reported structures which contain only one Mg2+ ion, surprisingly, two Mn2+ ions are found in the catalytic center of the AceK-Mn2+ structure. Our enzymatic assays demonstrate that AceK-Mn2+ showed higher phosphatase activity than AceK-Mg2+, whereas the kinase activity was relatively unaffected. We created mutants of AceK for all metal-coordinating residues. The phosphatase activities of these mutants were significantly impaired, suggesting the pivotal role of the binuclear (M1-M2) core in AceK phosphatase catalysis. Moreover, we have studied the interactions of Mn2+ and Mg2+ with wild-type and mutant AceK and found that the number of metal ions bound to AceK is in full agreement with the crystal structures. Combined with the enzymatic results, we demonstrate that AceK exhibits phosphatase activity in the presence of two, but not one, Mn2+ ions, similar to PPM phosphatases. Taken together, we suggest that metal ions help AceK to balance and fine tune its kinase and phosphatase activities.
- WNT-3A-induced β-catenin signaling does not require signaling through heterotrimeric G proteins. [Journal Article]
- JBJ Biol Chem 2019 Jun 24
- The network of wingless/int-1 (WNT)-induced signaling pathways includes β-catenin-dependent and -independent pathways. β-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-med…
The network of wingless/int-1 (WNT)-induced signaling pathways includes β-catenin-dependent and -independent pathways. β-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, β-catenin signaling is initiated through engagement of a Frizzled (FZD)/LDL-receptor-related protein 5/6 (LRP5/6) receptor complex. FZDs are G protein-coupled receptors, but the question whether heterotrimeric G proteins are involved in WNT/β-catenin signaling remains unanswered. Here, we investigate whether acute activation of WNT/β-catenin signaling by purified WNT-3A requires functional signaling through heterotrimeric G proteins. Using genome editing, we ablated expression of Gs/Golf/Gq/G11/G12/G13/Gz in HEK293 (ΔG7) cells, leaving the expression of pertussis toxin (PTX)-sensitive Gi/o proteins unchanged, to assess whether WNT-3A activates WNT/β-catenin signaling in wildtype and ΔG7 cells devoid of functional G protein signaling. We monitored WNT-3A-induced activation by detection of phosphorylation of LDL receptor-related protein 6 (LRP6), electrophoretic mobility shift of the phosphoprotein Dishevelled (DVL), β-catenin stabilization and dephosphorylation and TCF-dependent transcription. We found that purified, recombinant WNT-3A efficiently induces WNT/β-catenin signaling in ΔG7 cells in both the absence and presence of Gi/o-blocking PTX. Furthermore, cells completely devoid of G protein expression, so called Gα-depleted HEK293 cells, maintain responsiveness to WNT-3A with regard to the hallmarks of WNT/β-catenin signalling. These findings corroborate the concept that heterotrimeric G proteins are not required for this FZD- and DVL-mediated signaling branch. Our observations agree with previous results arguing for FZD conformation-dependent functional selectivity between DVL and heterotrimeric G proteins. In conclusion, WNT/β-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins.
- Mu-KRAS attenuates Hippo signaling pathway through PKCι to sustain the growth of pancreatic cancer. [Journal Article]
- JCJ Cell Physiol 2019 Jun 23
- The atypical protein kinase C isoform ι (PKCι) is upregulated, which cooperates with mutated KRAS (mu-KRAS) to promote the development of pancreatic cancers. However, the exact role of PKCι in KRAS-m…
The atypical protein kinase C isoform ι (PKCι) is upregulated, which cooperates with mutated KRAS (mu-KRAS) to promote the development of pancreatic cancers. However, the exact role of PKCι in KRAS-mediated pancreatic tumorigenesis is not fully defined. In the present study, we demonstrate that mu-KRAS upregulates and activates PKCι, accompanied by dephosphorylation of large tumor suppressor (LATS), a key member of the growth-inhibiting Hippo signaling pathway. As a result, Yes-associated protein 1 (YAP1; a transcriptional coactivator) is dephosphorylated and translocates to the nucleus, which promotes transcription of downstream target genes to sustain the transformed growth of pancreatic cancer cells. In contrast, when PKCι is suppressed by the chemical inhibitor or small-hairpin RNA, the levels of phosphorylated LATS and YAP1 are elevated and YAP1 is excluded from the nucleus, which enhances the susceptibility of pancreatic cancer cells harboring mu-KRAS to apoptosis. These findings shed new light on the mechanisms underlying the pancreatic tumorigenesis initiated by mu-KRAS, and suggest that the PKCι-YAP1 signaling may potentially be therapeutically targeted for restricting the growth and inducing apoptosis in pancreatic tumors expressing mu-KRAS.
- Muscarinic acetylcholine receptors regulate the dephosphorylation of eukaryotic translation elongation factor 2 in SNU-407 colon cancer cells. [Journal Article]
- BBBiochem Biophys Res Commun 2019 Jun 18
- Previously, we showed that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. However, the molecular mechanisms underlying this event are p…
Previously, we showed that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. However, the molecular mechanisms underlying this event are poorly understood. Here, we asked whether mAChRs modulate the activity of eukaryotic translation elongation factor 2 (eEF2), which controls ribosomal translocation during the peptide elongation step. When SNU-407 cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation at T56 was decreased in a dose- and time-dependent manner. The muscarinic antagonist atropine almost completely blocked this effect of carbachol, demonstrating that mAChRs specifically regulate eEF2 dephosphorylation. We also investigated the signaling pathways that connect mAChR stimulation to eEF2 dephosphorylation using chemical inhibitors. Treating cells with U0126, a potent MEK1/2 inhibitor, decreased carbachol-stimulated eEF2 dephosphorylation. In contrast, the mTORC1 inhibitor rapamycin did not have a significant effect on eEF2 dephosphorylation. We also found that the protein kinase C (PKC) inhibitor GF109203X substantially reduced eEF2 dephosphorylation. Together, our experimental data indicate that the MEK1/2-ERK1/2 pathway and the PKC pathway, but not the mTORC1-S6K1 pathway, are involved in mAChR-mediated eEF2 dephosphorylation.
- Role of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DNA damage response. [Review]
- CSCell Stress 2019 Feb 21; 3(3):70-85
- Maintenance of genome integrity is fundamental for cellular physiology. Our hereditary information encoded in the DNA is intrinsically susceptible to suffer variations, mostly due to the constant pre…
Maintenance of genome integrity is fundamental for cellular physiology. Our hereditary information encoded in the DNA is intrinsically susceptible to suffer variations, mostly due to the constant presence of endogenous and environmental genotoxic stresses. Genomic insults must be repaired to avoid loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental anomalies and tumorigenesis. To safeguard our genome, cells have evolved a series of mechanisms collectively known as the DNA damage response (DDR). This surveillance system regulates multiple features of the cellular response, including the detection of the lesion, a transient cell cycle arrest and the restoration of the broken DNA molecule. While the role of multiple kinases in the DDR has been well documented over the last years, the intricate roles of protein dephosphorylation have only recently begun to be addressed. In this review, we have compiled recent information about the function of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DDR, focusing mainly on their capacity to regulate the DNA damage checkpoint and the repair mechanism encompassed in the restoration of a DNA lesion.
- Phosphorylated lipid-conjugated oligonucleotide selectively anchors on cell membranes with high alkaline phosphatase expression. [Journal Article]
- NCNat Commun 2019 06 20; 10(1):2704
- Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjuga…
Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjugated oligonucleotides fail to distinguish among different cell membranes. Herein, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) is reported for alkaline phosphatase (ALP)-dependent cell membrane adhesion. In the absence of ALP, DNA-lipid-P with its poor hydrophobicity shows only weak interaction with cell membrane. However, in the presence of the highly expressed plasma membrane-associated ALP, DNA-lipid-P is converted to lipid-conjugated oligonucleotide (DNA-lipid) by enzymatic dephosphorylation. As a result of such conversion, the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P and is thus able to insert into cell membranes in situ. Accordingly, DNA-lipid-P enables selective anchoring on cell membranes with elevated ALP level. Since elevated ALP level is a critical index of some diseases and even cancers, DNA-lipid-P holds promise for cell membrane engineering and disease diagnostics at the molecular level.
- Arginine dephosphorylation propels spore germination in bacteria. [Journal Article]
- PNProc Natl Acad Sci U S A 2019 Jun 20
- Bacterial spores can remain dormant for years but possess the remarkable ability to germinate, within minutes, once nutrients become available. However, it still remains elusive how such instant awak…
Bacterial spores can remain dormant for years but possess the remarkable ability to germinate, within minutes, once nutrients become available. However, it still remains elusive how such instant awakening of cellular machineries is achieved. Utilizing Bacillus subtilis as a model, we show that YwlE arginine (Arg) phosphatase is crucial for spore germination. Accordingly, the absence of the Arg kinase McsB accelerated the process. Arg phosphoproteome of dormant spores uncovered a unique set of Arg-phosphorylated proteins involved in key biological functions, including translation and transcription. Consequently, we demonstrate that during germination, YwlE dephosphorylates an Arg site on the ribosome-associated chaperone Tig, enabling its association with the ribosome to reestablish translation. Moreover, we show that Arg dephosphorylation of the housekeeping σ factor A (SigA), mediated by YwlE, facilitates germination by activating the transcriptional machinery. Subsequently, we reveal that transcription is reinitiated at the onset of germination and its recommencement precedes that of translation. Thus, Arg dephosphorylation elicits the most critical stages of spore molecular resumption, placing this unusual post-translational modification as a major regulator of a developmental process in bacteria.
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- Neuronally Enriched RUFY3 Is Required for Caspase-Mediated Axon Degeneration. [Journal Article]
- NNeuron 2019 Jun 07
- Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy…
Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.