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1
Shen, Chwan-Li, Jannette M. Dufour, Jonathan M. Miranda, Gurvinder Kaur, Eunhee Chung, Latha Ramalingam, Naima Moustaid-Moussa, and Jay J. Cao. "Effect of Dietary Geranylgeraniol and Green Tea Polyphenols on Glucose Homeostasis, Bone Turnover Biomarkers, and Bone Microstructure in Obese Mice." International Journal of Molecular Sciences 24, no. 2 (January 4, 2023): 979. http://dx.doi.org/10.3390/ijms24020979.
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Previously, we demonstrated that the administration of either geranylgeraniol (GGOH) or green tea polyphenols (GTP) improved bone health. This study examined the combined effects of GGOH and GTP on glucose homeostasis in addition to bone remodeling in obese mice. We hypothesized that GGOH and GTP would have an additive or synergistic effect on improving glucose homeostasis and bone remodeling possibly in part via suppression of proinflammatory cytokines. Forty-eight male C57BL/6J mice were assigned to a high-fat diet (control), HFD + 400 mg GGOH/kg diet (GG), HFD + 0.5% GTP water (TP), or HFD + GGOH + GTP (GGTP) diet for 14 weeks. Results demonstrated that GTP supplementation improved glucose tolerance in obese mice. Neither GGOH nor GTP affected pancreas insulin or bone formation procollagen type I intact N-terminal, bone volume at the lumbar vertebrae, or bone parameters at the trabecular bone and cortical bone of the femur. There was an interactive effect for serum bone resorption collagen type 1 cross-linked C-telopeptide concentrations, resulting in no-GGOH and no-GTP groups having the highest values. GGOH increased trabecular number and decreased trabecular separation at the lumbar vertebrae. GTP increased trabecular thickness at lumbar vertebrae. The GG group produced the greatest connectivity density and the lowest structure model index. Only GTP, not GGOH, decreased adipokines concentrations (resistin, leptin, monocyte chemoattractant protein-1, and interleukin-6). In an obese male mouse model, individual GGOH and GTP supplementation improved glucose homeostasis, serum CTX, and trabecular microstructure of LV-4. However, the combined GGOH and GTP supplementation compromises such osteoprotective effects on serum CTX and trabecular bone of obese mice.
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Seccia, Roberta, Silvia De Santis, Maria A. Di Noia, Ferdinando Palmieri, Daniela V. Miniero, Raffaele Marmo, Eleonora Paradies, et al. "Citrate Regulates the Saccharomyces cerevisiae Mitochondrial GDP/GTP Carrier (Ggc1p) by Triggering Unidirectional Transport of GTP." Journal of Fungi 8, no. 8 (July 29, 2022): 795. http://dx.doi.org/10.3390/jof8080795.
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The yeast mitochondrial transport of GTP and GDP is mediated by Ggc1p, a member of the mitochondrial carrier family. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria in exchange for GDP generated in the matrix. ggc1Δ cells exhibit lower levels of GTP and increased levels of GDP in mitochondria, are unable to grow on nonfermentable substrates and lose mtDNA. Because in yeast, succinyl-CoA ligase produces ATP instead of GTP, and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space, Ggc1p is the only supplier of mitochondrial GTP required for the maturation of proteins containing Fe-S clusters, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. In this work, it was demonstrated that citrate is a regulator of purified and reconstituted Ggc1p by trans-activating unidirectional transport of GTP across the proteoliposomal membrane. It was also shown that the binding site of Ggc1p for citrate is different from the binding site for the substrate GTP. It is proposed that the citrate-induced GTP uniport (CIGU) mediated by Ggc1p is involved in the homeostasis of the guanine nucleotide pool in the mitochondrial matrix.
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Rodríguez-Fdez, Sonia, and Xosé R. Bustelo. "Rho GTPases in Skeletal Muscle Development and Homeostasis." Cells 10, no. 11 (November 2, 2021): 2984. http://dx.doi.org/10.3390/cells10112984.
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Rho guanosine triphosphate hydrolases (GTPases) are molecular switches that cycle between an inactive guanosine diphosphate (GDP)-bound and an active guanosine triphosphate (GTP)-bound state during signal transduction. As such, they regulate a wide range of both cellular and physiological processes. In this review, we will summarize recent work on the role of Rho GTPase-regulated pathways in skeletal muscle development, regeneration, tissue mass homeostatic balance, and metabolism. In addition, we will present current evidence that links the dysregulation of these GTPases with diseases caused by skeletal muscle dysfunction. Overall, this information underscores the critical role of a number of members of the Rho GTPase subfamily in muscle development and the overall metabolic balance of mammalian species.
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Khadilkar, Rohan J., Diana Rodrigues, Ridim Dadasaheb Mote, Arghyashree Roychowdhury Sinha, Vani Kulkarni, Srivathsa Subramanya Magadi, and Maneesha S. Inamdar. "ARF1–GTP regulates Asrij to provide endocytic control ofDrosophilablood cell homeostasis." Proceedings of the National Academy of Sciences 111, no. 13 (March 18, 2014): 4898–903. http://dx.doi.org/10.1073/pnas.1303559111.
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Mazhab-Jafari, Mohammad T., Christopher B. Marshall, Noboru Ishiyama, Jason Ho, Vanessa Di Palma, Vuk Stambolic, and Mitsuhiko Ikura. "An Autoinhibited Noncanonical Mechanism of GTP Hydrolysis by Rheb Maintains mTORC1 Homeostasis." Structure 20, no. 9 (September 2012): 1528–39. http://dx.doi.org/10.1016/j.str.2012.06.013.
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Chen, Hui-Jie, Na Li, Ye Luo, Yong-Liang Jiang, Cong-Zhao Zhou, Yuxing Chen, and Qiong Li. "The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis." Biochemical Journal 475, no. 7 (April 9, 2018): 1295–308. http://dx.doi.org/10.1042/bcj20180079.
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The second messenger c-di-GMP [bis-(3′-5′)-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian cGMP-regulated phosphodiesterases, Anabaenaadenylyl cyclases and Escherichia coli transcription activator FhlA) domain containing bifunctional enzyme DcpA (diguanylate cyclase and phosphodiesterase A) that catalyzes the synthesis and hydrolysis of c-di-GMP. Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5′-phosphoguanylyl-(3′-5′)-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments.
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Kristensson, Maria Alvarado. "The Game of Tubulins." Cells 10, no. 4 (March 28, 2021): 745. http://dx.doi.org/10.3390/cells10040745.
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Members of the tubulin superfamily are GTPases; the activities of GTPases are necessary for life. The members of the tubulin superfamily are the constituents of the microtubules and the γ-tubulin meshwork. Mutations in members of the tubulin superfamily are involved in developmental brain disorders, and tubulin activities are the target for various chemotherapies. The intricate functions (game) of tubulins depend on the activities of the GTP-binding domain of α-, β-, and γ-tubulin. This review compares the GTP-binding domains of γ-tubulin, α-tubulin, and β-tubulin and, based on their similarities, recapitulates the known functions and the impact of the γ-tubulin GTP-binding domain in the regulation of the γ-tubulin meshwork and cellular homeostasis.
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Kotelevets, Larissa, and Eric Chastre. "Rac1 Signaling: From Intestinal Homeostasis to Colorectal Cancer Metastasis." Cancers 12, no. 3 (March 12, 2020): 665. http://dx.doi.org/10.3390/cancers12030665.
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The small GTPase Rac1 has been implicated in a variety of dynamic cell biological processes, including cell proliferation, cell survival, cell-cell contacts, epithelial mesenchymal transition (EMT), cell motility, and invasiveness. These processes are orchestrated through the fine tuning of Rac1 activity by upstream cell surface receptors and effectors that regulate the cycling Rac1-GDP (off state)/Rac1-GTP (on state), but also through the tuning of Rac1 accumulation, activity, and subcellular localization by post translational modifications or recruitment into molecular scaffolds. Another level of regulation involves Rac1 transcripts stability and splicing. Downstream, Rac1 initiates a series of signaling networks, including regulatory complex of actin cytoskeleton remodeling, activation of protein kinases (PAKs, MAPKs) and transcription factors (NFkB, Wnt/β-catenin/TCF, STAT3, Snail), production of reactive oxygen species (NADPH oxidase holoenzymes, mitochondrial ROS). Thus, this GTPase, its regulators, and effector systems might be involved at different steps of the neoplastic progression from dysplasia to the metastatic cascade. After briefly placing Rac1 and its effector systems in the more general context of intestinal homeostasis and in wound healing after intestinal injury, the present review mainly focuses on the several levels of Rac1 signaling pathway dysregulation in colorectal carcinogenesis, their biological significance, and their clinical impact.
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Gumataotao, Natalie, K. P. Wasantha Lankathilaka, Brian Bennett, and Richard C. Holz. "The iron-type nitrile hydratase activator protein is a GTPase." Biochemical Journal 474, no. 2 (January 6, 2017): 247–58. http://dx.doi.org/10.1042/bcj20160884.
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The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing GTP to GDP with a kcat value of 1.2 × 10−3 s−1 and a Km value of 40 μM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes.
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Mori, Risa, and Takashi Toda. "The dual role of fission yeast Tbc1/cofactor C orchestrates microtubule homeostasis in tubulin folding and acts as a GAP for GTPase Alp41/Arl2." Molecular Biology of the Cell 24, no. 11 (June 2013): 1713–24. http://dx.doi.org/10.1091/mbc.e12-11-0792.
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Supplying the appropriate amount of correctly folded α/β-tubulin heterodimers is critical for microtubule dynamics. Formation of assembly-competent heterodimers is remarkably elaborate at the molecular level, in which the α- and β-tubulins are separately processed in a chaperone-dependent manner. This sequential step is performed by the tubulin-folding cofactor pathway, comprising a specific set of regulatory proteins: cofactors A–E. We identified the fission yeast cofactor: the orthologue of cofactor C, Tbc1. In addition to its roles in tubulin folding, Tbc1 acts as a GAP in regulating Alp41/Arl2, a highly conserved small GTPase. Of interest, the expression of GDP- or GTP-bound Alp41 showed the identical microtubule loss phenotype, suggesting that continuous cycling between these forms is important for its functions. In addition, we found that Alp41 interacts with Alp1D, the orthologue of cofactor D, specifically when in the GDP-bound form. Intriguingly, Alp1D colocalizes with microtubules when in excess, eventually leading to depolymerization, which is sequestered by co-overproducing GDP-bound Alp41. We present a model of the final stages of the tubulin cofactor pathway that includes a dual role for both Tbc1 and Alp1D in opposing regulation of the microtubule.
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Zolotarov, Yevgen, Chao Ma, Irene González-Recio, Serge Hardy, Gijs A. C. Franken, Noriko Uetani, Femke Latta, et al. "ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs." Cellular and Molecular Life Sciences 78, no. 13 (June 5, 2021): 5427–45. http://dx.doi.org/10.1007/s00018-021-03832-8.
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AbstractCyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like GTPase 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-β-synthase (CBS) domains. In silico modeling of the interaction between CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 and CNBH domains. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the endoplasmic reticulum, Golgi apparatus and the plasma membrane. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.
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Hildebrand, J. D., J. M. Taylor, and J. T. Parsons. "An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase." Molecular and Cellular Biology 16, no. 6 (June 1996): 3169–78. http://dx.doi.org/10.1128/mcb.16.6.3169.
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The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.
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Kriel, Allison, Alycia N. Bittner, Sok Ho Kim, Kuanqing Liu, Ashley K. Tehranchi, Winnie Y. Zou, Samantha Rendon, Rui Chen, Benjamin P. Tu, and Jue D. Wang. "Direct Regulation of GTP Homeostasis by (p)ppGpp: A Critical Component of Viability and Stress Resistance." Molecular Cell 48, no. 2 (October 2012): 231–41. http://dx.doi.org/10.1016/j.molcel.2012.08.009.
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Fiorentini, Carla, Paola Matarrese, Elisabetta Straface, Loredana Falzano, Alessia Fabbri, Gianfranco Donelli, Andrea Cossarizza, Patrice Boquet, and Walter Malorni. "Toxin-Induced Activation of Rho GTP-Binding Protein Increases Bcl-2 Expression and Influences Mitochondrial Homeostasis." Experimental Cell Research 242, no. 1 (July 1998): 341–50. http://dx.doi.org/10.1006/excr.1998.4057.
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García, Patricia, Rubén Celador, Jorge Pérez-Parrilla, and Yolanda Sánchez. "Fission Yeast Rho1p-GEFs: From Polarity and Cell Wall Synthesis to Genome Stability." International Journal of Molecular Sciences 23, no. 22 (November 11, 2022): 13888. http://dx.doi.org/10.3390/ijms232213888.
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Rho1p is a membrane-associated protein that belongs to the Rho family of small GTPases. These proteins coordinate processes such as actin remodelling and polarised secretion to maintain the shape and homeostasis of yeast cells. In response to extracellular stimuli, Rho1p undergoes conformational switching between a guanosine triphosphate (GTP)-bound active state and a guanosine diphosphate (GDP)-bound inactive state. Cycling is improved with guanine nucleotide exchange factor (GEF) activity necessary to activate signalling and GTPase activating protein (GAP) activity required for subsequent signal depletion. This review focuses on fission yeast Rho1p GEFs, Rgf1p, Rgf2p, and Rgf3p that belong to the family of DH-PH domain-containing Dbl-related GEFs. They are multi-domain proteins that detect biological signals that induce or inhibit their catalytic activity over Rho1p. Each of them activates Rho1p in different places and times. Rgf1p acts preferentially during polarised growth. Rgf2p is required for sporulation, and Rgf3p plays an essential function in septum synthesis. In addition, we outline the noncanonical roles of Rho1p-GEFs in genomic instability.
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Corre, Tanguy, Francisco J. Arjona, Caroline Hayward, Sonia Youhanna, Jeroen H. F. de Baaij, Hendrica Belge, Nadine Nägele, et al. "Genome-Wide Meta-Analysis Unravels Interactions between Magnesium Homeostasis and Metabolic Phenotypes." Journal of the American Society of Nephrology 29, no. 1 (November 1, 2017): 335–48. http://dx.doi.org/10.1681/asn.2017030267.
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Magnesium (Mg2+) homeostasis is critical for metabolism. However, the genetic determinants of the renal handling of Mg2+, which is crucial for Mg2+ homeostasis, and the potential influence on metabolic traits in the general population are unknown. We obtained plasma and urine parameters from 9099 individuals from seven cohorts, and conducted a genome-wide meta-analysis of Mg2+ homeostasis. We identified two loci associated with urinary magnesium (uMg), rs3824347 (P=4.4×10−13) near TRPM6, which encodes an epithelial Mg2+ channel, and rs35929 (P=2.1×10−11), a variant of ARL15, which encodes a GTP-binding protein. Together, these loci account for 2.3% of the variation in 24-hour uMg excretion. In human kidney cells, ARL15 regulated TRPM6-mediated currents. In zebrafish, dietary Mg2+ regulated the expression of the highly conserved ARL15 ortholog arl15b, and arl15b knockdown resulted in renal Mg2+ wasting and metabolic disturbances. Finally, ARL15 rs35929 modified the association of uMg with fasting insulin and fat mass in a general population. In conclusion, this combined observational and experimental approach uncovered a gene–environment interaction linking Mg2+ deficiency to insulin resistance and obesity.
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Brauner, C. J., and R. E. Weber. "Hydrogen ion titrations of the anodic and cathodic haemoglobin components of the European eel Anguilla anguilla." Journal of Experimental Biology 201, no. 17 (September 1, 1998): 2507–14. http://dx.doi.org/10.1242/jeb.201.17.2507.
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H+ titrations were conducted on the separated haemoglobin components of eel Anguilla anguilla in both the oxygenated and deoxygenated states. In anodic haemoglobin, the addition of GTP, and to a lesser extent C1-, increased the magnitude of the Haldane effect and shifted its maximum value into the in vivo pH range. Of the 22 histidine residues in the anodic component, only approximately seven were titratable, presumably the beta-chain residues at positions 41, 97, 109 and 146 (helical positions C7, FG4, G11 and HC3, respectively). In cathodic haemoglobin, a small negative Haldane effect was observed at pH values between 6.8 and 8.5 which disappeared in the presence of GTP (molar ratio 3:1 GTP:haemoglobin tetramer). GTP had virtually no effect on the buffer value at fixed oxygenation status, and the lowest buffer value was observed at in vivo pH values. No titratable histidine residues were observed in the cathodic component, indicating that all 14 histidines in this component are buried. We conclude that the anodic component, which constitutes two-thirds of the haemoglobin in the eel, plays the predominant role in CO2 transport and pH homeostasis in vivo.
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Chang, Jan-Gowth, Ni Tien, Yi-Chih Chang, Meng-Liang Lin, and Shih-Shun Chen. "Oxidative Stress-Induced Unscheduled CDK1–Cyclin B1 Activity Impairs ER–Mitochondria-Mediated Bioenergetic Metabolism." Cells 10, no. 6 (May 21, 2021): 1280. http://dx.doi.org/10.3390/cells10061280.
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Targeting the activities of endoplasmic reticulum (ER)–mitochondrial-dependent metabolic reprogramming is considered one of the most promising strategies for cancer treatment. Here, we present biochemical subcellular fractionation, coimmunoprecipitation, gene manipulation, and pharmacologic evidence that induction of mitochondria-localized phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)–cyclin B1 complexes by apigenin in nasopharyngeal carcinoma (NPC) cells impairs the ER–mitochondrial bioenergetics and redox regulation of calcium (Ca++) homeostasis through suppressing the B cell lymphoma 2 (BCL-2)/BCL-2/B-cell lymphoma-extra large (BCL-xL)-modulated anti-apoptotic and metabolic functions. Using a specific inducer, inhibitor, or short hairpin RNA for acid sphingomyelinase (ASM) demonstrated that enhanced lipid raft-associated ASM activity confers alteration of the lipid composition of lipid raft membranes, which leads to perturbation of protein trafficking, and induces formation of p110α free p85α–unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 complexes in the lipid raft membranes, causing disruption of phosphatidylinositol 3-kinase (PI3K)−protein kinase B (Akt)−GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated signaling, thus triggering the p-CDK1 (Thr 161))–cyclin B1-mediated BCL-2 (Thr 69/Ser 87)/BCL-xL (Ser 62) phosphorylation and accompanying impairment of ER–mitochondria-regulated bioenergetic, redox, and Ca++ homeostasis. Inhibition of apigenin-induced reactive oxygen species (ROS) generation by a ROS scavenger N-acetyl-L-cysteine blocked the lipid raft membrane localization and activation of ASM and formation of ceramide-enriched lipid raft membranes, returned PI3K−Akt−GTP-Rac1-modulated CDK1–cyclin B1 activity, and subsequently restored the BCL-2/BCL-xL-regulated ER–mitochondrial bioenergetic activity. Thus, this study reveals a novel molecular mechanism of the pro-apoptotic activity of ASM controlled by oxidative stress to modulate the ER–mitochondrial bioenergetic metabolism, as well as suggests the disruption of CDK1–cyclin B1-mediated BCL-2/BCL-xL oncogenic activity by triggering oxidative stress−ASM-induced PI3K−Akt−GTP-Rac1 inactivation as a therapeutic approach for NPC.
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Sadacca, L. Amanda, Joanne Bruno, Jennifer Wen, Wenyong Xiong, and Timothy E. McGraw. "Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs." Molecular Biology of the Cell 24, no. 16 (August 15, 2013): 2544–57. http://dx.doi.org/10.1091/mbc.e13-02-0103.
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Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.
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Lee, Moonsup, Yoo-Seok Hwang, Jaeho Yoon, Jian Sun, Adam Harned, Kunio Nagashima, and Ira O. Daar. "Developmentally regulated GTP-binding protein 1 modulates ciliogenesis via an interaction with Dishevelled." Journal of Cell Biology 218, no. 8 (July 3, 2019): 2659–76. http://dx.doi.org/10.1083/jcb.201811147.
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Cilia are critical for proper embryonic development and maintaining homeostasis. Although extensively studied, there are still significant gaps regarding the proteins involved in regulating ciliogenesis. Using the Xenopus laevis embryo, we show that Dishevelled (Dvl), a key Wnt signaling scaffold that is critical to proper ciliogenesis, interacts with Drg1 (developmentally regulated GTP-binding protein 1). The loss of Drg1 or disruption of the interaction with Dvl reduces the length and number of cilia and displays defects in basal body migration and docking to the apical surface of multiciliated cells (MCCs). Moreover, Drg1 morphants display abnormal rotational polarity of basal bodies and a decrease in apical actin and RhoA activity that can be attributed to disruption of the protein complex between Dvl and Daam1, as well as between Daam1 and RhoA. These results support the concept that the Drg1–Dvl interaction regulates apical actin polymerization and stability in MCCs. Thus, Drg1 is a newly identified partner of Dvl in regulating ciliogenesis.
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Miaw, Shi-Chuen, and Wei-Yao Chin. "Adenylate kinase 4 is critical to the function of classically activated macrophage." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 79.17. http://dx.doi.org/10.4049/jimmunol.210.supp.79.17.
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Abstract Macrophage plays a crucial role in the front line of host defense against pathogens. Classically activated macrophages (M1), induced by IFN-g and LPS, highly express inflammatory cytokines and contribute to inflammatory processes. By contrast, alternatively activated macrophages (M2) are induced by IL-4/IL-13, produce IL-10, and display anti-inflammatory activity. Adenylate kinase 4 (Ak4), an enzyme that transfers phosphate group among ATP/GTP, AMP, and ADP, is a key modulator of ATP. Ak4 is involved in maintaining the homeostasis of cellular nucleotides which is essential for cellular function. We observed Ak4 is preferentially expressed in M1 macrophages compared to M2 macrophages. Whether Ak4 is critical for M1 macrophage function remains elusive. Here we demonstrated that Ak4 maintained ATP homeostasis, and was critical for ROS production, glycolysis, and bactericidal ability in M1 macrophages. Moreover, Ak4 promoted the expression of inflammatory genes, including Il1b, Il6, Tnfa, Nos2, Nox2and Hif1a, in M1 macrophages via Hif1a and AMPK. However, Ak4 deficiency did not affect the development of murine immune cells. Taken together, our data depict a potential mechanism linking nucleotide homeostasis and the function of M1 macrophage. Taiwan Ministry of Science and Technology (111-2320-B-002-068-MY3)
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Wolf, B. A., J. R. Colca, J. Turk, J. Florholmen, and M. L. McDaniel. "Regulation of Ca2+ homeostasis by islet endoplasmic reticulum and its role in insulin secretion." American Journal of Physiology-Endocrinology and Metabolism 254, no. 2 (February 1, 1988): E121—E136. http://dx.doi.org/10.1152/ajpendo.1988.254.2.e121.
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Changes in intracellular Ca2+ concentrations have a major role in the regulation of insulin secretion by islet beta-cells. It has recently become apparent that the endoplasmic reticulum plays a prominent role in the regulation of intracellular Ca2+ concentrations under basal conditions and during insulin secretion. This review describes biochemical properties of the endoplasmic reticulum that contribute to intracellular Ca2+ homeostasis including 1) an ATP-dependent Ca2+ uptake pump associated with a Ca2+-ATPase located in the endoplasmic reticulum; 2) Ca2+ release from the endoplasmic reticulum induced by the second messengers inositol trisphosphate and arachidonic acid as well as the guanine nucleotide GTP; and 3) a Ca2+ sequestration mechanism localized to the endoplasmic reticulum that is regulated by glucose 6-phosphate and glucose-6-phosphatase. The hypothesis is developed that these biochemical mechanisms participate in the regulation of intracellular Ca2+ concentrations and represent central intracellular events involved in the first phase of glucose-induced insulin secretion.
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Gaca, Anthony O., Pavel Kudrin, Cristina Colomer-Winter, Jelena Beljantseva, Kuanqing Liu, Brent Anderson, Jue D. Wang, et al. "From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis." Journal of Bacteriology 197, no. 18 (June 29, 2015): 2908–19. http://dx.doi.org/10.1128/jb.00324-15.
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ABSTRACTThe bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. InEnterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolaseE. faecalisRel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation inFirmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEfsynthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEfalso efficiently utilized GMP to form GMP 3′-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity ofE. faecalisenzymes involved in GTP biosynthesis and, to a lesser extent, transcription ofrrnBbyEscherichia coliRNA polymerase. Activation ofE. coliRelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEfwas activated only by ppGpp. Furthermore, enzymatic activity of RelQEfis insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of “long” RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp.IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ ofEnterococcus faecalis(RelQEf), we found that, in addition to (p)ppGpp, RelQEfis an efficient producer of pGpp (GMP 3′-diphosphate).In vitroanalysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEfand suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.
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Lee, Tsung-Lin, Shyang-Guang Wang, Wen-Ling Chan, Ching-Hsiao Lee, Tian-Shung Wu, Meng-Liang Lin, and Shih-Shun Chen. "Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells." Cells 9, no. 2 (February 5, 2020): 371. http://dx.doi.org/10.3390/cells9020371.
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The specialized cholesterol/sphingolipid-rich membrane domains termed lipid rafts are highly dynamic in the cancer cells, which rapidly assemble effector molecules to form a sorting platform essential for oncogenic signaling transduction in response to extra- or intracellular stimuli. Density-based membrane flotation, subcellular fractionation, cell surface biotinylation, and co-immunoprecipitation analyses of bichalcone analog ((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4)-treated cancer cells showed dissociation between GRP78 and p85α conferring the recruitment of PTEN to lipid raft membranes associated with p85α. Ectopic expression of GRP78 could overcome induction of lipid raft membrane-associated p85α–unphosphorylated PTEN complex formation and suppression of GRP78−PI3K−Akt−GTP-Rac1-mediated and GRP78-regulated PERK−Nrf2 antioxidant pathway and cancer cell invasion by TSWU-BR4. Using specific inducer, inhibitor, or short hairpin RNA for ASM demonstrated that induction of the lipid raft membrane localization and activation of ASM by TSWU-BR4 is responsible for perturbing homeostasis of cholesterol and ceramide levels in the lipid raft and ER membranes, leading to alteration of GRP78 membrane trafficking and subsequently inducing p85α–unphosphorylated PTEN complex formation, causing disruption of GRP78−PI3K−Akt−GTP-Rac1-mediated signal and ER membrane-associated GRP78-regulated oxidative stress balance, thus inhibiting cancer cell invasion. The involvement of the enrichment of ceramide to lipid raft membranes in inhibition of NF-κB-mediated MMP-2 expression was confirmed through attenuation of NF-κB activation using C2-ceramide, NF-κB specific inhibitors, ectopic expression of NF-κB p65, MMP-2 promoter-driven luciferase, and NF-κB-dependent reporter genes. In conclusion, localization of ASM in the lipid raft membranes by TSWU-BR4 is a key event for initiating formation of ceramide-enriched lipid raft membrane platforms, which causes delocalization of GRP78 from the lipid raft and ER membranes to the cytosol and formation of p85α–unphosphorylated PTEN complexes to attenuate the GRP78-regulated oxidative stress balance and GRP78−p85α−Akt−GTP-Rac1−NF-κB−MMP-2-mediated cancer cell invasion.
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Fujisawa, Koichi, Maina Wakazaki, Aya Matsuzaki, Toshihiko Matsumoto, Naoki Yamamoto, Takafumi Noma, and Taro Takami. "Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism." International Journal of Molecular Sciences 23, no. 8 (April 13, 2022): 4316. http://dx.doi.org/10.3390/ijms23084316.
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The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg2+ GTP + AMP ⇌ Mg2+ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.
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Covian, Raul, Lanelle Edwards, Yi He, Geumsoo Kim, Carly Houghton, Rodney L. Levine, and Robert S. Balaban. "Energy homeostasis is a conserved process: Evidence from Paracoccus denitrificans’ response to acute changes in energy demand." PLOS ONE 16, no. 11 (November 8, 2021): e0259636. http://dx.doi.org/10.1371/journal.pone.0259636.
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Paracoccus denitrificans is a model organism for the study of oxidative phosphorylation. We demonstrate a very high respiratory capacity compared to mitochondria when normalizing to cytochrome aa3 content even in the absence of alternative terminal oxidases. To gain insight into conserved mechanisms of energy homeostasis, we characterized the metabolic response to K+ reintroduction. A rapid 3-4-fold increase in respiration occurred before substantial cellular K+ accumulation followed by a sustained increase of up to 6-fold that persisted after net K+ uptake stopped. Proton motive force (Δp) was slightly higher upon addition of K+ with ΔpH increasing and compensating for membrane potential (ΔΨ) depolarization. Blocking the F0F1-ATP synthase (Complex V) with venturicidin revealed that the initial K+-dependent respiratory activation was primarily due to K+ influx. However, the ability to sustain an increased respiration rate was partially dependent on Complex V activity. The 6-fold stimulation of respiration by K+ resulted in a small net reduction of most cytochromes, different from the pattern observed with chemical uncoupling and consistent with balanced input and utilization of reducing equivalents. Metabolomics showed increases in glycolytic and TCA cycle intermediates together with a decrease in basic amino acids, suggesting an increased nitrogen mobilization upon K+ replenishment. ATP and GTP concentrations increased after K+ addition, indicating a net increase in cellular potential energy. Thus, K+ stimulates energy generation and utilization resulting in an almost constant Δp and increased high-energy phosphates during large acute and steady state changes in respiration. The specific energy consuming processes and signaling events associated with this simultaneous activation of work and metabolism in P. denitrificans remain unknown. Nevertheless, this homeostatic behavior is very similar to that observed in mitochondria in tissues when cellular energy requirements increase. We conclude that the regulation of energy generation and utilization to maintain homeostasis is conserved across the prokaryote/eukaryote boundary.
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Reed, Colbie J., Geoffrey Hutinet, and Valérie de Crécy-Lagard. "Comparative Genomic Analysis of the DUF34 Protein Family Suggests Role as a Metal Ion Chaperone or Insertase." Biomolecules 11, no. 9 (August 27, 2021): 1282. http://dx.doi.org/10.3390/biom11091282.
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Members of the DUF34 (domain of unknown function 34) family, also known as the NIF3 protein superfamily, are ubiquitous across superkingdoms. Proteins of this family have been widely annotated as “GTP cyclohydrolase I type 2” through electronic propagation based on one study. Here, the annotation status of this protein family was examined through a comprehensive literature review and integrative bioinformatic analyses that revealed varied pleiotropic associations and phenotypes. This analysis combined with functional complementation studies strongly challenges the current annotation and suggests that DUF34 family members may serve as metal ion insertases, chaperones, or metallocofactor maturases. This general molecular function could explain how DUF34 subgroups participate in highly diversified pathways such as cell differentiation, metal ion homeostasis, pathogen virulence, redox, and universal stress responses.
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Anthony, Sajitha A., Anika L. Burrell, Matthew C. Johnson, Krisna C. Duong-Ly, Yin-Ming Kuo, Jacqueline C. Simonet, Peter Michener, Andrew Andrews, Justin M. Kollman, and Jeffrey R. Peterson. "Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations." Molecular Biology of the Cell 28, no. 20 (October 2017): 2600–2608. http://dx.doi.org/10.1091/mbc.e17-04-0263.
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Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear, but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas guanosine triphosphate (GTP) promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and nonassembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.
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Purvis, Jeremy E., Manash S. Chatterjee, Lawrence F. Brass, and Scott L. Diamond. "A molecular signaling model of platelet phosphoinositide and calcium regulation during homeostasis and P2Y1 activation." Blood 112, no. 10 (November 15, 2008): 4069–79. http://dx.doi.org/10.1182/blood-2008-05-157883.
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Abstract To quantify how various molecular mechanisms are integrated to maintain platelet homeostasis and allow responsiveness to adenosine diphosphate (ADP), we developed a computational model of the human platelet. Existing kinetic information for 77 reactions, 132 fixed kinetic rate constants, and 70 species was combined with electrochemical calculations, measurements of platelet ultrastructure, novel experimental results, and published single-cell data. The model accurately predicted: (1) steady-state resting concentrations for intracellular calcium, inositol 1,4,5-trisphosphate, diacylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol 4,5-bisphosphate; (2) transient increases in intracellular calcium, inositol 1,4,5-trisphosphate, and Gq-GTP in response to ADP; and (3) the volume of the platelet dense tubular system. A more stringent test of the model involved stochastic simulation of individual platelets, which display an asynchronous calcium spiking behavior in response to ADP. Simulations accurately reproduced the broad frequency distribution of measured spiking events and demonstrated that asynchronous spiking was a consequence of stochastic fluctuations resulting from the small volume of the platelet. The model also provided insights into possible mechanisms of negative-feedback signaling, the relative potency of platelet agonists, and cell-to-cell variation across platelet populations. This integrative approach to platelet biology offers a novel and complementary strategy to traditional reductionist methods.
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Yu, Jing, Luis E. Gimenez, Ciria C. Hernandez, Yiran Wu, Ariel H. Wein, Gye Won Han, Kyle McClary, et al. "Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding." Science 368, no. 6489 (April 23, 2020): 428–33. http://dx.doi.org/10.1126/science.aaz8995.
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The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte–stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)–independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein–coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.
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Tan, Chuanting, Yulei Du, Lianhui Zhu, Shuaiyang Jing, Jingkai Gao, Yi Qian, Xihua Yue, and Intaek Lee. "KDEL Receptor Trafficking to the Plasma Membrane Is Regulated by ACBD3 and Rab4A-GTP." Cells 12, no. 7 (April 4, 2023): 1079. http://dx.doi.org/10.3390/cells12071079.
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KDEL receptor-1 maintains homeostasis in the early secretory pathway by capturing and retrieving ER chaperones to the ER during heavy secretory activity. Unexpectedly, a fraction of the receptor is also known to reside in the plasma membrane (PM), although it is largely unknown exactly how the KDEL receptor gets exported from the Golgi and travels to the PM. We have previously shown that a Golgi scaffolding protein (ACBD3) facilitates KDEL receptor localization at the Golgi via the regulating cargo wave-induced cAMP/PKA-dependent signaling pathway. Upon endocytosis, surface-expressed KDEL receptor undergoes highly complex itineraries through the Golgi and the endo-lysosomal compartments, where the endocytosed receptor utilizes Rab14A- and Rab11A-positive recycling endosomes and clathrin-decorated tubulovesicular carriers. In this study, we sought to investigate the mechanism through which the KDEL receptor gets exported from the Golgi en route to the PM. We report here that ACBD3 depletion results in greatly increased trafficking of KDEL receptor to the PM via Rab4A-positive tubular carriers emanating from the Golgi. Expression of constitutively activated Rab4A mutant (Q72L) increases the surface expression of KDEL receptor up to 2~3-fold, whereas Rab4A knockdown or the expression of GDP-locked Rab4A mutant (S27N) inhibits KDEL receptor targeting of the PM. Importantly, KDELR trafficking from the Golgi to the PM is independent of PKA- and Src kinase-mediated mechanisms. Taken together, these results reveal that ACBD3 and Rab4A play a key role in regulating KDEL receptor trafficking to the cell surface.
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Makino, Asami, Françoise Hullin-Matsuda, Motohide Murate, Mitsuhiro Abe, Nario Tomishige, Mitsunori Fukuda, Shizuya Yamashita, et al. "Acute accumulation of free cholesterol induces the degradation of perilipin 2 and Rab18-dependent fusion of ER and lipid droplets in cultured human hepatocytes." Molecular Biology of the Cell 27, no. 21 (November 2016): 3293–304. http://dx.doi.org/10.1091/mbc.e15-10-0730.
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Dysregulated hepatic cholesterol homeostasis with free cholesterol accumulation in the liver is relevant to the pathogenesis of nonalcoholic steatohepatitis, contributing to the chronicity of liver toxicity. Here we examined the effect of free cholesterol accumulation on the morphology and biochemical properties of lipid droplets (LDs) in cultured hepatocytes. Acute free cholesterol accumulation induced the fusion of LDs, followed by degradation of the coat protein of LDs, perilipin 2 (PLIN2; also called adipophilin or adipose differentiation–related protein), and association of apolipoprotein B 100 (ApoB 100) to LDs. The degradation of PLIN2 was inhibited by inhibitors of ubiquitination, autophagy, and protein synthesis. The results indicate that association of ApoB 100 with LDs is dependent on the activity of low–molecular weight GTP-binding protein Rab18 and highlight the role of LDs as targets of free cholesterol toxicity in hepatocytes.
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Voorberg, Jan, Mariska G. Rondaij, Karina A. Gijzen, Ruben Bierings, Erica Sellink, Mar Fernandez-Borja, and Jan A. van Mourik. "The Guanine Exchange Factor RalGDS Is Involved in Regulated Exocytosis of Weibel-Palade Bodies from Endothelial Cells." Blood 106, no. 11 (November 16, 2005): 3688. http://dx.doi.org/10.1182/blood.v106.11.3688.3688.
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Abstract Endothelial cells contribute to vascular homeostasis and mediate pathophysiological responses to hypoxia-induced injury and inflammatory events. To ensure rapid responses to vascular perturbation endothelial cells contain intracellular storage pools for inflammatory mediators and pro-thrombotic compounds. One of the best characterized storage granules within endothelial cells are the Weibel-Palade bodies (WPB), rod-shaped organelles that contains P-selectin, Von Willebrand Factor (VWF), interleukin-8 (IL-8) and a number of other proteins with diverse biological activities. Agonist-induced triggering of heterotrimeric G protein coupled receptors (GPCR) present on endothelial cells promote exocytosis of WPB. We have previously shown that the small GTP binding protein RalA is involved in thrombin induced exocytosis of Weibel-Palade bodies. Exocytosis of Weibel-Palade bodies was found to coincide with the activation of RalA in response to thrombin. More recently, we have shown that cAMP-raising stimuli such as epinephrine also coincide with the activation of RalA. Consistent with these findings constitutively active RalG23V was capable of inducing release of WPB from endothelial cells. Small GTPases are activated by guanine exchange factors (GEFs) that induce GDP release thereby enhancing GTP binding to the small GTPase. RalGDS is a widely expressed GEF for Ral that has recently been implicated in cytoskeletal rearrangements that result from activation of GPCRs. Here, we investigated whether RalGDS is involved in thrombin- and/or epinephrine-induced exocytosis of WPB from endothelial cells. First, we showed by RT-PCR that human endothelial cells express RalGDS. Overexpression of a GFP-tagged variant of RalGDS in endothelial cells reduces the number of WPB in endothelial cells suggesting that RalGDS can promote exocytosis of these subcellular organelles. To investigate whether endogenously synthesized RalGDS plays a role in exocytosis of WPBs we designed short hairpin RNAs that acts as small interfering RNA (siRNA). Co-expression of siRalGDS and GFP-RalGDS in heterologously transfected 293 cells markedly reduced expression levels of GFP-RalGDS. Subsequently, we addressed the effect of siRalGDS on exocytosis of WPB in endothelial cells. Knockdown of endogenous RalGDS using siRNA prevented thrombin-induced release of WPBs. Expression of siRalGDS also interfered with WPB release in response to epinephrine. These results show that knock down of RalGDS interferes with exocytosis of WPB in endothelial cells. A dominant negative RalGDS variant, RalGDSΔGEF, lacking its catalytic exchange domain, was subsequently introduced in endothelial cells. As expected, no release of WPB was observed in endothelial cells expressing RalGDSΔGEF. Remarkably, both thrombin- and epinephrine-induced exocytosis were impaired in cells expressing RalGDSΔGEF. Together, these findings indicate that the Ral-specific guanine exchange factor RalGDS is involved in exocytosis of WPB from endothelial cells.
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Lo, C., J. Ferrier, H. C. Tenenbaum, and C. A. G. McCulloch. "Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells." Biochemistry and Cell Biology 73, no. 7-8 (July 1, 1995): 535–44. http://dx.doi.org/10.1139/o95-059.
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The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of ~30% in suspended (i.e., round) cells and ~60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with B APTA–AM and by depletion of Cl−, indicating the role of calcium-regulated K+ and Cl− efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (~0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na+-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl−-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl− that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.Key words: cell volume, pH, osteoblast, G proteins, actin.
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Rufanova, Victoriya A., Anna Alexanian, Tammo Ostendorf, Dirk Bokemeyer, Simon Prosser, Bradley Miller, and Andrey Sorokin. "Endothelin signaling via guanine exchange factor C3G in renal glomerular mesangial cellsThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research." Canadian Journal of Physiology and Pharmacology 88, no. 8 (August 2010): 808–16. http://dx.doi.org/10.1139/y10-056.
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The guanine nucleotide exchange factor C3G is one of the mediators of endothelin-1 (ET-1) intracellular signaling cascades and is vital for kidney development and homeostasis. The aim of the current study was to analyze the specificity of ET-1-induced signaling via C3G in rat glomerular mesangial cells (GMC) and to investigate the biological significance of C3G during mesangioproliferative glomerulonephritis. In GMC, C3G expression was increased (1) in vivo after induction of the anti-Thy1 model of glomerulonephritis and (2) in cell culture experiments after fetal bovine serum incubation. To examine the consequences of C3G up-regulation, adenovirus-mediated gene transfer of C3G into cultured glomerular cells was done, and the GTP loading of the small G proteins Rap1 and R-Ras was analyzed. Overexpression of C3G in mesangial cells resulted in enhanced activation of Rap1, but failed to affect the GTP-bound status of R-Ras in ET-1-stimulated cells. C3G overexpression led to significant changes in GMC spreading and migration patterns in response to ET-1 stimulation and increased stress fiber formation, which was mimicked by Rap1A overexpression. Together, these findings suggest (1) the existence of regulatory mechanisms resulting in disease-related up-regulation of C3G in GMC and (2) that an increase in the C3G protein level may contribute to the resolution stage of mesangioproliferative glomerulonephritis by reducing GMC sensitivity to ET-1, modulating cellular motility, and actin dynamics.
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Morris, Elizabeth R., and Ian A. Taylor. "The missing link: allostery and catalysis in the anti-viral protein SAMHD1." Biochemical Society Transactions 47, no. 4 (July 11, 2019): 1013–27. http://dx.doi.org/10.1042/bst20180348.
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Abstract Vertebrate protein SAMHD1 (sterile-α-motif and HD domain containing protein 1) regulates the cellular dNTP (2′-deoxynucleoside-5′-triphosphate) pool by catalysing the hydrolysis of dNTP into 2′-deoxynucleoside and triphosphate products. As an important regulator of cell proliferation and a key player in dNTP homeostasis, mutations to SAMHD1 are implicated in hypermutated cancers, and germline mutations are associated with Chronic Lymphocytic Leukaemia and the inflammatory disorder Aicardi–Goutières Syndrome. By limiting the supply of dNTPs for viral DNA synthesis, SAMHD1 also restricts the replication of several retroviruses, such as HIV-1, and some DNA viruses in dendritic and myeloid lineage cells and resting T-cells. SAMHD1 activity is regulated throughout the cell cycle, both at the level of protein expression and post-translationally, through phosphorylation. In addition, allosteric regulation further fine-tunes the catalytic activity of SAMHD1, with a nucleotide-activated homotetramer as the catalytically active form of the protein. In cells, GTP and dATP are the likely physiological activators of two adjacent allosteric sites, AL1 (GTP) and AL2 (dATP), that bridge monomer–monomer interfaces to stabilise the protein homotetramer. This review summarises the extensive X-ray crystallographic, biophysical and molecular dynamics experiments that have elucidated important features of allosteric regulation in SAMHD1. We present a comprehensive mechanism detailing the structural and protein dynamics components of the allosteric coupling between nucleotide-induced tetramerization and the catalysis of dNTP hydrolysis by SAMHD1.
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Bonventre, J. V. "Phospholipase A2 and signal transduction." Journal of the American Society of Nephrology 3, no. 2 (August 1992): 128–50. http://dx.doi.org/10.1681/asn.v32128.
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Phospholipases A2 (PLA2) comprise a family of enzymes that hydrolyze the acyl bond at the sn-2 position of phospholipids to generate free fatty acids and lysophospholipids. Different forms of PLA2 are involved in digestion, inflammation, and intercellular and intracellular signal transduction. The sn-2 position of phospholipids in mammalian cells is enriched in arachidonic acid, the precursor of eicosanoids, which have diverse physiologic and pathophysiologic effects on the kidney and other organs. Thus, the regulation of PLA2 activity has important implications for kidney function. PLA2 regulation involves: calcium, pH, protein kinases, GTP-binding proteins, inhibitory and activating proteins, metabolic product inhibition, and transcriptional control. The various roles of arachidonic acid and cyclooxygenase, lipoxygenase, and cytochrome P450 mono-oxygenase products of arachidonic acid metabolism, as intracellular messengers, in the regulation of membrane channel activities, intracellular enzyme activities, cellular calcium homeostasis, mitogenesis, differentiation, cytokine and early response gene expression are discussed.
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Bandorowicz-Pikuła, J., M. Danieluk, A. Wrzosek, R. Buś, R. Buchet, and S. Pikuła. "Annexin VI: an intracellular target for ATP." Acta Biochimica Polonica 46, no. 3 (September 30, 1999): 801–12. http://dx.doi.org/10.18388/abp.1999_4152.
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Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.
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Wilkinson, Beverley, Jocelyn S. Downey, and Christopher E. Rudd. "T-cell signalling and immune system disorders." Expert Reviews in Molecular Medicine 7, no. 29 (December 19, 2005): 1–29. http://dx.doi.org/10.1017/s1462399405010264.
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T-cell receptor (TCR) engagement initiates intracellular signalling cascades that lead to T-cell proliferation, cytokine production and differentiation into effector cells. These cascades comprise an array of protein-tyrosine kinases, phosphatases, GTP-binding proteins and adaptor proteins that regulate generic and specialised functions. The integration of these signals is essential for the normal development, homeostasis and function of T cells. Defects in a single mediator can produce T cells that are unable to participate fully in an immune response and/or that mount an inappropriate response, which leads to immunodeficiency, autoimmunity or leukaemia/lymphomas. This review highlights some of the key players in T-cell signalling and their involvement in the development of various clinical disease states. Some of these immune-specific signalling proteins are attractive potential targets in the development of therapies to augment T-cell responses to antigen or tumours, and to treat immune cell disorders.
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Pradhan, Rashmita, Phuong A. Ngo, Luz d. C. Martínez-Sánchez, Markus F. Neurath, and Rocío López-Posadas. "Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases." Cells 10, no. 1 (January 4, 2021): 66. http://dx.doi.org/10.3390/cells10010066.
Full textAbstract:
Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.
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Navarro-Lérida, Inmaculada, Miguel Sánchez-Álvarez, and Miguel Ángel del Pozo. "Post-Translational Modification and Subcellular Compartmentalization: Emerging Concepts on the Regulation and Physiopathological Relevance of RhoGTPases." Cells 10, no. 8 (August 5, 2021): 1990. http://dx.doi.org/10.3390/cells10081990.
Full textAbstract:
Cells and tissues are continuously exposed to both chemical and physical stimuli and dynamically adapt and respond to this variety of external cues to ensure cellular homeostasis, regulated development and tissue-specific differentiation. Alterations of these pathways promote disease progression—a prominent example being cancer. Rho GTPases are key regulators of the remodeling of cytoskeleton and cell membranes and their coordination and integration with different biological processes, including cell polarization and motility, as well as other signaling networks such as growth signaling and proliferation. Apart from the control of GTP–GDP cycling, Rho GTPase activity is spatially and temporally regulated by post-translation modifications (PTMs) and their assembly onto specific protein complexes, which determine their controlled activity at distinct cellular compartments. Although Rho GTPases were traditionally conceived as targeted from the cytosol to the plasma membrane to exert their activity, recent research demonstrates that active pools of different Rho GTPases also localize to endomembranes and the nucleus. In this review, we discuss how PTM-driven modulation of Rho GTPases provides a versatile mechanism for their compartmentalization and functional regulation. Understanding how the subcellular sorting of active small GTPase pools occurs and what its functional significance is could reveal novel therapeutic opportunities.
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Kim, Hyungdong, Nour Ghaddar, Laleh Ebrahimi Ghahnavieh, Shuo Wang, Kwang-Jin Cho, Atsuo Sasaki, and Antonis E. Koromilas. "Abstract A022: Translation initiation factor 2B (eIF2B) stimulates mutant KRAS function in cancer." Molecular Cancer Research 21, no. 5_Supplement (May 1, 2023): A022. http://dx.doi.org/10.1158/1557-3125.ras23-a022.
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Abstract KRAS mutations appear with high frequency in colorectal, lung and pancreatic cancers, which are the three leading causes of new cancer deaths worldwide. Mutant KRAS is preferentially bound to GTP resulting in continuous cell proliferation. Mutant KRAS exposes cells to oncogenic forms of stress (i.e. genotoxic, metabolic, proteostatic stress), which disrupt proliferation and tissue homeostasis. To cope with stress, cells engage pro-adaptive mechanisms, which act in favor of mutant KRAS to transform cells. An important adaptation mechanism to stress acts at the level of mRNA translation and involves the functional interplay between the translation initiator factors eIF2 and eIF2B. Phosphorylated eIF2 mediates a translational and transcriptional reprogramming to promote adaptation under stress, a process that is antagonized by the guanine exchange function (GEF) of eIF2B. We demonstrate the physical interaction between mutant KRAS and eIF2B by mass spectrometry. Using genetic approaches, we show that eIF2B is required for the survival and proliferation of tumor cells with KRAS mutations via the stimulation of MAPK signaling. We also show that eIF2B contributes to increased resistance of tumor cells to pharmacological inhibition of mutant KRAS forms. Genetic inactivation of eIF2B promotes the formation of mutant KRAS-GDP complexes whereas its pharmacological stimulation facilitates mutant KRAS-GTP complex formation in tumor cells; this data supports a potential GEF function for eIF2B towards mutant KRAS. Cell imaging experiments provide strong evidence for the implication of eIF2B in the association of mutant KRAS with the plasma membrane of tumor cells. Our findings reveal a stimulatory role of eIF2B in mutant KRAS signaling and provide a previously unidentified link between mutant KRAS and mRNA translation with implications in the growth and treatment of cancers with KRAS mutations. Citation Format: Hyungdong Kim, Nour Ghaddar, Laleh Ebrahimi Ghahnavieh, Shuo Wang, Kwang-Jin Cho, Atsuo Sasaki, Antonis E. Koromilas. Translation initiation factor 2B (eIF2B) stimulates mutant KRAS function in cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A022.
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Guo, Yusong, and Adam D. Linstedt. "COPII–Golgi protein interactions regulate COPII coat assembly and Golgi size." Journal of Cell Biology 174, no. 1 (July 3, 2006): 53–63. http://dx.doi.org/10.1083/jcb.200604058.
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Under experimental conditions, the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum (ER), involving a rapid phase of growth followed by a return to steady state, but the mechanisms that control growth are unknown. Quantification of coat protein complex (COP) II assembly revealed a dramatic up-regulation at exit sites driven by increased levels of Golgi proteins in the ER. Analysis in a permeabilized cell assay indicated that up-regulation of COPII assembly occurred in the absence GTP hydrolysis and any cytosolic factors other than the COPII prebudding complex Sar1p–Sec23p–Sec24p. Remarkably, acting via a direct interaction with Sar1p, increased expression of the Golgi enzyme N-acetylgalactosaminyl transferase-2 induced increased COPII assembly on the ER and an overall increase in the size of the Golgi apparatus. These results suggest that direct interactions between Golgi proteins exiting the ER and COPII components regulate ER exit, providing a variable exit rate mechanism that ensures homeostasis of the Golgi apparatus.
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Li, Lequn, Rebecca Greenwald, Esther M. Lafuente, Dimitrios Tzachanis, Alla Berezovskaya, Gordon J. Freeman, Arlene H. Sharpe, and Vassiliki A. Boussiotis. "Rap1-GTP Promotes the Generation of Regulatory T Cells in Vivo." Blood 104, no. 11 (November 16, 2004): 110. http://dx.doi.org/10.1182/blood.v104.11.110.110.
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Abstract Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.
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Balcerzak, Marcin, Eva Hamade, Le Zhang, Slawomir Pikula, Gérard Azzar, Jacqueline Radisson, Joanna Bandorowicz-Pikula, and Rene Buchet. "The roles of annexins and alkaline phosphatase in mineralization process." Acta Biochimica Polonica 50, no. 4 (December 31, 2003): 1019–38. http://dx.doi.org/10.18388/abp.2003_3629.
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In this review the roles of specific proteins during the first step of mineralization and nucleation are discussed. Mineralization is initiated inside the extracellular organelles-matrix vesicles (MVs). MVs, containing relatively high concentrations of Ca2+ and inorganic phosphate (Pi), create an optimal environment to induce the formation of hydroxyapatite (HA). Special attention is given to two families of proteins present in MVs, annexins (AnxAs) and tissue-nonspecific alkaline phosphatases (TNAPs). Both families participate in the formation of HA crystals. AnxAs are Ca2+ - and lipid-binding proteins, which are involved in Ca2+ homeostasis in bone cells and in extracellular MVs. AnxAs form calcium ion channels within the membrane of MVs. Although the mechanisms of ion channel formation by AnxAs are not well understood, evidence is provided that acidic pH or GTP contribute to this process. Furthermore, low molecular mass ligands, as vitamin A derivatives, can modulate the activity of MVs by interacting with AnxAs and affecting their expression. AnxAs and other anionic proteins are also involved in the crystal nucleation. The second family of proteins, TNAPs, is associated with Pi homeostasis, and can hydrolyse a variety of phosphate compounds. ATP is released in the extracellular matrix, where it can be hydrolyzed by TNAPs, ATP hydrolases and nucleoside triphosphate (NTP) pyrophosphohydrolases. However, TNAP is probably not responsible for ATP-dependent Ca2+/phosphate complex formation. It can hydrolyse pyrophosphate (PPi), a known inhibitor of HA formation and a byproduct of NTP pyrophosphohydrolases. In this respect, antagonistic activities of TNAPs and NTP pyrophosphohydrolases can regulate the mineralization process.
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Deshpande, Pratima, Karnail Singh, Cornelia Weyand, and Jorg Goronzy. "TCR tuning by homeostatic cytokines — implication for autoimmunity (101.16)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 101.16. http://dx.doi.org/10.4049/jimmunol.186.supp.101.16.
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Abstract In animal models, lymphopenia and the associated function of homeostatic cytokines (HC) set the stage for autoimmunity. This model is relevant for rheumatoid arthritis (RA) which has defects in T cell homeostasis due to a failure in DNA repair and which responds to JAK inhibition. We have previously shown that RA T cells have a hyperreactive ERK response which impairs the induction of anergy. Here, we examined whether HC tune TCR activation thresholds by calibrating the ERK rheostat. RA T cells exhibited increased levels of phosphorylated STAT3 and STAT5 indicating in vivo activity of IL-21/IL6 and IL-7/IL-15. Incubation of healthy donor T cells with IL-7, IL15 or IL21 prior to anti-CD3/CD28 stimulation augmented ERK activation and expression of activation markers (CD69, CD40L) and cytokines (TNFα, IFNγ and IL-17). Preincubation with IL-1β or TNFα did not exert an effect. In DR4 healthy donors, HC priming facilitated responses to self-antigens such as citrullinated vimentin peptide. The effect of HC lasted for less than 3 hours and was sensitive to PI3K inhibition suggesting a non-transcriptional PI3K mediated priming of the ERK pathway. HC dose titration showed a bimodal distribution in pERK consistent with an on-off switch characteristic of a positive feedback loop. Allosteric binding of RAS-GTP to SOS is known to induce such an on-off switch. Indeed, HC incubation increased active RAS. We propose that HC lower the threshold for autoimmune responses by priming of SOS.
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Basu, Arnab, and Mee-Ngan F. Yap. "Disassembly of theStaphylococcus aureushibernating 100S ribosome by an evolutionarily conserved GTPase." Proceedings of the National Academy of Sciences 114, no. 39 (September 11, 2017): E8165—E8173. http://dx.doi.org/10.1073/pnas.1709588114.
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The bacterial hibernating 100S ribosome is a poorly understood form of the dimeric 70S particle that has been linked to pathogenesis, translational repression, starvation responses, and ribosome turnover. In the opportunistic pathogenStaphylococcus aureusand most other bacteria, hibernation-promoting factor (HPF) homodimerizes the 70S ribosomes to form a translationally silent 100S complex. Conversely, the 100S ribosomes dissociate into subunits and are presumably recycled for new rounds of translation. The regulation and disassembly of the 100S ribosome are largely unknown because the temporal abundance of the 100S ribosome varies considerably among different bacterial phyla. Here, we identify a universally conserved GTPase (HflX) as a bona fide dissociation factor of theS. aureus100S ribosome. The expression levelshpfandhflXare coregulated by general stress and stringent responses in a temperature-dependent manner. While all tested guanosine analogs stimulate the splitting activity of HflX on the 70S ribosome, only GTP can completely dissociate the 100S ribosome. Our results reveal the antagonistic relationship of HPF and HflX and uncover the key regulators of 70S and 100S ribosome homeostasis that are intimately associated with bacterial survival.
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Morgan, Niamh E., Meritxell B. Cutrona, and Jeremy C. Simpson. "Multitasking Rab Proteins in Autophagy and Membrane Trafficking: A Focus on Rab33b." International Journal of Molecular Sciences 20, no. 16 (August 12, 2019): 3916. http://dx.doi.org/10.3390/ijms20163916.
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Autophagy (particularly macroautophagy) is a bulk degradation process used by eukaryotic cells in order to maintain adequate energy levels and cellular homeostasis through the delivery of long-lived proteins and organelles to the lysosome, resulting in their degradation. It is becoming increasingly clear that many of the molecular requirements to fulfil autophagy intersect with those of conventional and unconventional membrane trafficking pathways. Of particular interest is the dependence of these processes on multiple members of the Rab family of small GTP binding proteins. Rab33b is a protein that localises to the Golgi apparatus and has suggested functions in both membrane trafficking and autophagic processes. Interestingly, mutations in the RAB33B gene have been reported to cause the severe skeletal disorder, Smith–McCort Dysplasia; however, the molecular basis for Rab33b in this disorder remains to be determined. In this review, we focus on the current knowledge of the participation of Rab33b and its interacting partners in membrane trafficking and macroautophagy, and speculate on how its function, and dysfunction, may contribute to human disease.
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Inamdar, Arati A., Anathbandhu Chaudhuri, and Janis O’Donnell. "The Protective Effect of Minocycline in a Paraquat-Induced Parkinson's Disease Model inDrosophilais Modified in Altered Genetic Backgrounds." Parkinson's Disease 2012 (2012): 1–16. http://dx.doi.org/10.1155/2012/938528.
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Epidemiological studies link the herbicide paraquat to increased incidence of Parkinson's disease (PD). We previously reported thatDrosophilaexposed to paraquat recapitulate PD symptoms, including region-specific degeneration of dopaminergic neurons. Minocycline, a tetracycline derivative, exerts ameliorative effects in neurodegenerative disease models, includingDrosophila. We investigated whether our environmental toxin-based PD model could contribute to an understanding of cellular and genetic mechanisms of minocycline action and whether we could assess potential interference with these drug effects in altered genetic backgrounds. Cofeeding of minocycline with paraquat prolonged survival, rescued mobility defects, blocked generation of reactive oxygen species, and extended dopaminergic neuron survival, as has been reported previously for a genetic model of PD inDrosophila. We then extended this study to identify potential interactions of minocycline with genes regulating dopamine homeostasis that might modify protection against paraquat and found that deficits in GTP cyclohydrolase adversely affect minocycline rescue. We further performed genetic studies to identify signaling pathways that are necessary for minocycline protection against paraquat toxicity and found that mutations in theDrosophilagenes that encode c-Jun N-terminal kinase (JNK) and Akt/Protein kinase B block minocycline rescue.
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Caruso, Marie-Elaine, Sarah Jenna, Marion Bouchecareilh, David L. Baillie, Daniel Boismenu, Dalia Halawani, Martin Latterich, and Eric Chevet. "GTPase-Mediated Regulation of the Unfolded Protein Response in Caenorhabditis elegans Is Dependent on the AAA+ ATPase CDC-48." Molecular and Cellular Biology 28, no. 13 (May 5, 2008): 4261–74. http://dx.doi.org/10.1128/mcb.02252-07.
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ABSTRACT When endoplasmic reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the unfolded protein response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, and ATF-6) mediate the reestablishment of ER functions but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions, such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, among other, ER-related functions: the small GTP-binding proteins of the RAS superfamily. To this end, we used transgenic UPR reporter Caenorhabditis elegans strains as a model to specifically silence small-GTPase expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA+ ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.