15 results on '"Shears, Stephen B."'
Search Results
2. InsP₇ is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics
- Author
-
Sahu, Soumyadip, Wang, Zhenzhen, Jiao, Xinfu, Gu, Chunfang, Jork, Nikolaus, Wittwer, Christopher, Li, Xingyao, Hostachy, Sarah, Fiedler, Dorothea, Wang, Huanchen, Jessen, Henning J., Kiledjian, Megerditch, and Shears, Stephen B.
- Published
- 2020
3. Control of XPR1-dependent cellular phosphate efflux by InsP₈ is an exemplar for functionally-exclusive inositol pyrophosphate signaling
- Author
-
Li, Xingyao, Gu, Chunfang, Hostachy, Sarah, Sahu, Soumyadip, Wittwer, Christopher, Jessen, Henning J., Fiedler, Dorothea, Wang, Huanchen, and Shears, Stephen B.
- Published
- 2020
4. Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress
- Author
-
Sung, Eui Jae, Ryuda, Masasuke, Matsumoto, Hitoshi, Uryu, Outa, Ochiai, Masanori, Cook, Molly E., Yi, Na Young, Wang, Huanchen, Putney, James W., Bird, Gary S., Shears, Stephen B., and Hayakawa, Yoichi
- Published
- 2017
5. KO of 5-InsP₇ kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype
- Author
-
Gu, Chunfang, Nguyen, Hoai-Nghia, Ganini, Douglas, Chen, Zhaowei, Jessen, Henning J., Gu, Zhen, Wang, Huanchen, and Shears, Stephen B.
- Published
- 2017
6. Dephosphorylation of 2,3-Bisphosphoglycerate by MIPP Expands the Regulatory Capacity of the Rapoport-Luebering Glycolytic Shunt
- Author
-
Cho, Jaiesoon, King, Jason S., Qian, Xun, Harwood, Adrian J., and Shears, Stephen B.
- Published
- 2008
- Full Text
- View/download PDF
7. Telomere Maintenance by Intracellular Signals: New Kid on the Block?
- Author
-
Shears, Stephen B.
- Published
- 2005
8. Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line.
- Author
-
Chunfang Gu, Juan Liu, Xiaojing Liu, Haibo Zhang, Ji Luo, Huanchen Wang, Locasale, Jason W., and Shears, Stephen B.
- Subjects
PENTOSE phosphate pathway ,CELL lines ,INOSITOL ,CURCUMIN ,METABOLIC regulation ,PROTEIN kinases - Abstract
Identification of common patterns of cancer metabolic reprogramming could assist the development of new therapeutic strategies. Recent attention in this field has focused on identifying and targeting signal transduction pathways that interface directly with major metabolic control processes. In the current study we demonstrate the importance of signaling by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to the metabolism and proliferation of the HCT116 colonic tumor cell line. We observed reciprocal cross talk between PPIP5K catalytic activity and glucose metabolism, and we show that CRISPR-mediated PPIP5K deletion suppresses HCT116 cell proliferation in glucose-limited culture conditions that mimic the tumor cell microenvironment. We conducted detailed, global metabolomic analyses of wild-type and PPIP5K knockout (KO) cells by measuring both steady-state metabolite levels and by performing isotope tracing experiments. We attribute the growth-impaired phenotype to a specific reduction in the supply of precursor material for de novo nucleotide biosynthesis from the one carbon serine/glycine pathway and the pentose phosphate pathway. We identify two enzymatic control points that are inhibited in the PPIP5K KO cells: serine hydroxymethyltransferase and phosphoribosyl pyrophosphate synthetase, a known downstream target of AMP-regulated protein kinase, which we show is noncanonically activated independently of adenine nucleotide status. Finally, we show the proliferative defect in PPIP5K KO cells can be significantly rescued either by addition of inosine monophosphate or a nucleoside mixture or by stable expression of PPIP5K activity. Overall, our data describe multiple, farreaching metabolic consequences for metabolic supervision by PPIP5Ks in a tumor cell line. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
9. Control of XPR1-dependent cellular phosphate efflux by InsP8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling.
- Author
-
Xingyao Li, Chunfang Gu, Hostachy, Sarah, Soumyadip Sahu, Wittwer, Christopher, Jessen, Henning J., Fiedler, Dorothea, Huanchen Wang, and Shears, Stephen B.
- Subjects
CYTOLOGY ,ISOTHERMAL titration calorimetry ,METABOLIC regulation ,ORGANIC compounds ,PHOSPHATES - Abstract
Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP
8 ). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP8 synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP8 ) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP8 levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8 ; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP8 to the XPR1 N-terminus (Kd = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification. [ABSTRACT FROM AUTHOR]- Published
- 2020
- Full Text
- View/download PDF
10. Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress.
- Author
-
Uryu, Outa, Eui Jae Sung, Huanchen Wang, Shears, Stephen B., Masasuke Ryuda, Hitoshi Matsumoto, Yoichi Hayakawa, Masanori Ochiai, Cook, Molly E., Na Young Yi, Putney, James W., and Bird, Gary S.
- Subjects
CYTOKINES ,DROSOPHILA ,GROWTH factors ,GENETICS of longevity ,PEPTIDES - Abstract
A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca
2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
11. KO of 5-InsP7 kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype.
- Author
-
Chunfang Gu, Hoai-Nghia Nguyen, Ganini, Douglas, Zhaowei Chen, Jessen, Henning J., Zhen Gu, Huanchen Wang, and Shears, Stephen B.
- Subjects
COLON cancer ,CRISPRS ,RNA interference ,GENETIC overexpression ,CELL cycle ,P53 antioncogene ,GENE expression ,CELL metabolism - Abstract
The inositol pyrophosphates 5-InsP
7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8 (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7 ] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7 ] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7 -generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
12. Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport–Luebering glycolytic shunt.
- Author
-
Jaiesoon Cho, King, Jason S., Xun Qian, Harwood, Adrian J., and Shears, Stephen B.
- Subjects
CHEMICAL reactions ,ERYTHROCYTES ,HEMOGLOBIN polymorphisms ,PROTEIN kinases ,BIOCHEMISTRY - Abstract
The Rapoport-Luebering glycolytic bypass comprises evolutionarily conserved reactions that generate and dephosphorylate 2,3- bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyostelium, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase. Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mippi expression in Dictyosteliurn, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37°C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4°C. revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
13. InsP 7 is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics.
- Author
-
Sahu S, Wang Z, Jiao X, Gu C, Jork N, Wittwer C, Li X, Hostachy S, Fiedler D, Wang H, Jessen HJ, Kiledjian M, and Shears SB
- Subjects
- Acid Anhydride Hydrolases genetics, HEK293 Cells, Humans, Phosphotransferases (Phosphate Group Acceptor) genetics, Phosphotransferases (Phosphate Group Acceptor) metabolism, RNA Caps genetics, RNA Stability, RNA, Messenger genetics, Acid Anhydride Hydrolases metabolism, Inositol Phosphates metabolism, RNA Caps metabolism, RNA, Messenger metabolism
- Abstract
Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP
7 (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7 inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP7 levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7 synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7 analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7 levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging., Competing Interests: The authors declare no competing interest.- Published
- 2020
- Full Text
- View/download PDF
14. Control of XPR1-dependent cellular phosphate efflux by InsP 8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling.
- Author
-
Li X, Gu C, Hostachy S, Sahu S, Wittwer C, Jessen HJ, Fiedler D, Wang H, and Shears SB
- Subjects
- Biological Transport, HEK293 Cells, Humans, Phosphates metabolism, Phosphotransferases (Phosphate Group Acceptor) genetics, Phosphotransferases (Phosphate Group Acceptor) metabolism, Receptors, G-Protein-Coupled genetics, Receptors, Virus genetics, Signal Transduction, Xenotropic and Polytropic Retrovirus Receptor, Phosphatidylinositol Phosphates metabolism, Receptors, G-Protein-Coupled metabolism, Receptors, Virus metabolism
- Abstract
Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP
8 ). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP8 synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP8 ) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP8 levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8 ; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP8 to the XPR1 N-terminus ( Kd = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification., Competing Interests: The authors declare no competing interest.- Published
- 2020
- Full Text
- View/download PDF
15. KO of 5-InsP 7 kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype.
- Author
-
Gu C, Nguyen HN, Ganini D, Chen Z, Jessen HJ, Gu Z, Wang H, and Shears SB
- Subjects
- CRISPR-Cas Systems, Cell Line, Tumor, Colonic Neoplasms metabolism, G1 Phase Cell Cycle Checkpoints genetics, Gene Knockout Techniques, Glycolysis genetics, Glycolysis physiology, HCT116 Cells, HEK293 Cells, Humans, Mitochondria metabolism, Phosphotransferases (Phosphate Group Acceptor) metabolism, Signal Transduction, Adenosine Triphosphate metabolism, Cyclin-Dependent Kinase Inhibitor p21 biosynthesis, Inositol Phosphates metabolism, Phosphotransferases (Phosphate Group Acceptor) genetics, Tumor Suppressor Protein p53 biosynthesis
- Abstract
The inositol pyrophosphates 5-InsP
7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8 (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7 ] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7 ] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7 -generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N 2-( m -trifluorobenzyl), N 6-( p -nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)- Published
- 2017
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.