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Cells Feb 2020Oocyte maturation is a process that occurs in the ovaries, where an immature oocyte resumes meiosis to attain competence for normal fertilization after... (Review)
Review
Oocyte maturation is a process that occurs in the ovaries, where an immature oocyte resumes meiosis to attain competence for normal fertilization after ovulation/spawning. In starfish, the hormone 1-methyladenine binds to an unidentified receptor on the plasma membrane of oocytes, inducing a conformational change in the heterotrimeric GTP-binding protein α-subunit (Gα), so that the α-subunit binds GTP in exchange of GDP on the plasma membrane. The GTP-binding protein βγ-subunit (Gβγ) is released from Gα, and the released Gβγ activates phosphatidylinositol-3 kinase (PI3K), followed by the target of rapamycin kinase complex2 (TORC2) and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-dependent phosphorylation of serum- and glucocorticoid-regulated kinase (SGK) of ovarian oocytes. Thereafter, SGK activates Na/H exchanger (NHE) to increase the intracellular pH (pHi) from ~6.7 to ~6.9. Moreover, SGK phosphorylates Cdc25 and Myt1, thereby inducing the de-phosphorylation and activation of cyclin B-Cdk1, causing germinal vesicle breakdown (GVBD). Both pHi increase and GVBD are required for spindle assembly at metaphase I, followed by MI arrest at pHi 6.9 until spawning. Due to MI arrest or SGK-dependent pHi control, spawned oocytes can be fertilized normally.
Topics: Adenine; Animals; Female; Fertilization; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Hydrogen-Ion Concentration; Immediate-Early Proteins; Metaphase; Oocytes; Oogenesis; Ovary; Phosphorylation; Protein Serine-Threonine Kinases; Receptors, G-Protein-Coupled; Starfish
PubMed: 32092921
DOI: 10.3390/cells9020476 -
Trends in Pharmacological Sciences Jul 2018Until the late 1990s, class A G protein-coupled receptors (GPCRs) were believed to function as monomers. Indirect evidence that they might internalize or even signal as... (Review)
Review
Until the late 1990s, class A G protein-coupled receptors (GPCRs) were believed to function as monomers. Indirect evidence that they might internalize or even signal as dimers has emerged, along with proof that class C GPCRs are obligatory dimers. Crystal structures of GPCRs and their much larger binding partners were consistent with the idea that two receptors might engage a single G protein, GRK, or arrestin. However, recent biophysical, biochemical, and structural evidence invariably suggests that a single GPCR binds G proteins, GRKs, and arrestins. Here we review existing evidence of the stoichiometry of GPCR interactions with signal transducers and discuss potential biological roles of class A GPCR oligomers, including proposed homo- and heterodimers.
Topics: Animals; Arrestins; GTP-Binding Proteins; Humans; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 29739625
DOI: 10.1016/j.tips.2018.04.002 -
Frontiers in Cell and Developmental... 2018Rab7 - or in yeast, Ypt7p - governs membrane trafficking in the late endocytic and autophagic pathways. Rab7 also regulates mitochondrion-lysosome contacts, the sites of... (Review)
Review
Rab7 - or in yeast, Ypt7p - governs membrane trafficking in the late endocytic and autophagic pathways. Rab7 also regulates mitochondrion-lysosome contacts, the sites of mitochondrial fission. Like all Rab GTPases, Rab7 cycles between an "active" GTP-bound form that binds downstream effectors - e.g., the HOPS and retromer complexes and the dynactin-binding Rab-interacting lysosomal protein (RILP) - and an "inactive" GDP-bound form that cannot bind effectors. Accessory proteins regulate the nucleotide binding state of Rab7: guanine nucleotide exchange factors (GEFs) stimulate exchange of bound GDP for GTP, resulting in Rab7 activation, whereas GTPase activating proteins (GAPs) boost Rab7's GTP hydrolysis activity, thereby inactivating Rab7. This review will discuss the GEF and GAPs that control Rab7 nucleotide binding, and thus regulate Rab7's activity in endolysosomal trafficking and autophagy. It will also consider how bacterial pathogens manipulate Rab7 nucleotide binding to support intracellular invasion and immune evasion.
PubMed: 30333976
DOI: 10.3389/fcell.2018.00129 -
Frontiers in Molecular Biosciences 2022AtYchF1 is an unconventional G-protein in that exhibits relaxed nucleotide-binding specificity. The bindings between AtYchF1 and biomolecules including GTP, ATP, and...
AtYchF1 is an unconventional G-protein in that exhibits relaxed nucleotide-binding specificity. The bindings between AtYchF1 and biomolecules including GTP, ATP, and 26S rRNA have been reported. In this study, we demonstrated the binding of AtYchF1 to ppGpp in addition to the above molecules. AtYchF1 is a cytosolic protein previously reported as a negative regulator of both biotic and abiotic stresses while the accumulation of ppGpp in the cytoplasm induces retarded plant growth and development. By co-crystallization, pull-down experiments, and hydrolytic biochemical assays, we demonstrated the binding and hydrolysis of ppGpp by AtYchF1. ppGpp inhibits the binding of AtYchF1 to ATP, GTP, and 26S rRNA. The ppGpp hydrolyzing activity of AtYchF1 failed to be activated by AtGAP1. The AtYchF1-ppGpp co-crystal structure suggests that ppGpp might prevent His from executing nucleotide hydrolysis. In addition, upon the binding of ppGpp, the conformation between the TGS and helical domains of AtYchF1 changes. Such structural changes probably influence the binding between AtYchF1 and other molecules such as 26S rRNA. Since YchF proteins are conserved among different kingdoms of life, the findings advance the knowledge on the role of AtYchF1 in regulating nucleotide signaling as well as hint at the possible involvement of YchF proteins in regulating ppGpp level in other species.
PubMed: 36533075
DOI: 10.3389/fmolb.2022.1061350 -
FEBS Letters Apr 1987Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP-binding proteins (G-proteins) as defined by Hughes [(1983) Febs... (Review)
Review
Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP-binding proteins (G-proteins) as defined by Hughes [(1983) Febs Lett. 164, 1-8]. How tubulin is structurally related to G-proteins is not known. We use a synthesis of sequence comparisons between tubulin, other G-proteins, and ADP/ATP-binding proteins and topological arguments to identify potential regions involved in nucleotide binding. We propose that the nucleotide-binding domain in the beta-subunit of tubulin is an alpha/beta structure derived from amino acid residues approximately 60-300. Five peptide sequences are identified which we suggest exist as 'loops' that extend from beta-strands and connect alpha-helices in this structure. We argue that GDP binds to four of the five loops in an Mg2+-independent manner while GTP binds in an Mg2+-dependent manner to a different combination of four loops. We propose that this switch between loops upon GTP binding induces a conformational change essential for microtubule assembly.
Topics: Amino Acid Sequence; Binding Sites; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Models, Molecular; Protein Conformation; Tubulin
PubMed: 3106086
DOI: 10.1016/0014-5793(87)80061-3 -
Scientific Reports Jun 2018Microtubule plus end-binding protein, EB1 is a key regulator of microtubule dynamics. Auto-inhibitory interaction in EB1 has previously been shown to inhibit its ability...
Microtubule plus end-binding protein, EB1 is a key regulator of microtubule dynamics. Auto-inhibitory interaction in EB1 has previously been shown to inhibit its ability to bind to microtubules and regulate microtubule dynamics. However, the factors that promote its microtubule regulatory activity by over-coming the auto-inhibition are less known. Here, we show that GTP plays a critical role in promoting the microtubule-targeting activity of EB1 by suppressing its auto-inhibition. Our biophysical data demonstrate that GTP binds to EB1 at a distinct site in its conserved N-terminal domain. Detailed analyses reveal that GTP-binding suppresses the intra-molecular inhibitory interaction between the globular N-terminus and the C-terminal coiled-coil domain. We further show that mutation of the GTP-binding site residues in N-terminus weakens the affinity for GTP, but also for the C-terminus, indicating overlapping binding sites. Confocal imaging and biochemical analysis reveal that EB1 localization on the microtubules is significantly increased upon mutations of the GTP-binding site residues. The results demonstrate a unique role of GTP in facilitating EB1 interaction with the microtubules by relieving its intra-molecular inhibition. They also implicate that GTP-binding may regulate the functions of EB1 on the cellular microtubules.
Topics: Amino Acid Sequence; Amino Acids; Binding Sites; Guanosine Triphosphate; Humans; Microtubule-Associated Proteins; Microtubules; Mutation; Protein Domains
PubMed: 29955158
DOI: 10.1038/s41598-018-28056-y -
The Journal of Biological Chemistry Dec 2023MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer...
MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1's EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1's EF-hands bind Ca with dissociation constants (K) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca whereas the other EF-hand binds Ca in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.
Topics: Humans; GTP Phosphohydrolases; Guanosine Triphosphate; Kinesins; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; rho GTP-Binding Proteins
PubMed: 37949220
DOI: 10.1016/j.jbc.2023.105441 -
BMC Microbiology Feb 2011Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they...
BACKGROUND
Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they participate in spore formation, stress adaptation, ribosome assembly and chromosomal partitioning. This study was undertaken to investigate the biochemical and physiological characteristics of Obg in Mycobacterium tuberculosis, which causes tuberculosis in humans.
RESULTS
We overexpressed M. tuberculosis Obg in Escherichia coli and then purified the protein. This protein binds to, hydrolyzes and is phosphorylated with GTP. An anti-Obg antiserum, raised against the purified Obg, detects a 55 kDa protein in immunoblots of M. tuberculosis extracts. Immunoblotting also discloses that cultured M. tuberculosis cells contain increased amounts of Obg in the late log phase and in the stationary phase. Obg is also associated with ribosomes in M. tuberculosis, and it is distributed to all three ribosomal fractions (30 S, 50 S and 70 S). Finally, yeast two-hybrid analysis reveals that Obg interacts with the stress protein UsfX, indicating that M. tuberculosis Obg, like other bacterial Obgs, is a stress related protein.
CONCLUSIONS
Although its GTP-hydrolyzing and phosphorylating activities resemble those of other bacterial Obg homologues, M. tuberculosis Obg differs from them in these respects: (a) preferential association with the bacterial membrane; (b) association with all three ribosomal subunits, and (c) binding to the stress protein UsfX, rather than to RelA. Generation of mutant alleles of Obg of M. tuberculosis, and their characterization in vivo, may provide additional insights regarding its role in this important human pathogen.
Topics: Bacterial Proteins; DNA, Bacterial; Escherichia coli; GTP-Binding Proteins; Membrane Proteins; Mycobacterium tuberculosis; Phosphorylation; Protein Binding; Ribosomes; Two-Hybrid System Techniques
PubMed: 21352546
DOI: 10.1186/1471-2180-11-43 -
Small GTPases May 2020The small GTPase Rab7 is the main regulator of membrane trafficking at late endosomes. This small GTPase regulates endosome-to-trans Golgi Network trafficking of sorting... (Review)
Review
The small GTPase Rab7 is the main regulator of membrane trafficking at late endosomes. This small GTPase regulates endosome-to-trans Golgi Network trafficking of sorting receptors, membrane fusion of late endosomes to lysosomes, and autophagosomes to lysosomes during autophagy. Rab7, like all Rab GTPases, binds downstream effectors coordinating several divergent pathways. How cells regulate these interactions and downstream functions is not well understood. Recent evidence suggests that Rab7 function can be modulated by the combination of several post-translational modifications that facilitate interactions with one effector while preventing binding to another one. In this review, we discuss recent data on how phosphorylation, palmitoylation and ubiquitination modulate the ability of this small GTPase to orchestrate membrane trafficking at the late endosomes.
Topics: Humans; Lipoylation; Phosphorylation; Protein Processing, Post-Translational; Ubiquitination; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins
PubMed: 29099291
DOI: 10.1080/21541248.2017.1387686 -
Scientific Reports Nov 2017Rab5 is a small monomeric GTPase that mediates protein trafficking during endocytosis. Inactivation of Rab5 by GTP hydrolysis causes a conformational change that masks...
Rab5 is a small monomeric GTPase that mediates protein trafficking during endocytosis. Inactivation of Rab5 by GTP hydrolysis causes a conformational change that masks binding sites on its "switch regions" from downstream effectors. The p85 subunit of phosphatidylinositol 3-kinase (PI3K) is a GTPase activating protein (GAP) towards Rab5. Whereas p85 can bind with both Rab5-GTP and Rab5-GDP, the PI3K catalytic subunit p110β binds only Rab5-GTP, suggesting it interacts with the switch regions. Thus, the GAP functions of the catalytic arginine finger (from p85) and switch region stabilization (from p110β) may be provided by both proteins, acting together. To identify the Rab5 residues involved in binding p110β, residues in the Rab5 switch regions were mutated. A stabilized recombinant p110 protein, where the p85-iSH2 domain was fused to p110 (alpha or beta) was used in binding experiments. Eleven Rab5 mutants, including E80R and H83E, showed reduced p110β binding. The Rab5 binding site on p110β was also resolved through mutation of p110β in its Ras binding domain, and includes residues I234, E238 and Y244. This is a second region within p110β important for Rab5 binding. The Rab5-GTP:p110β interaction may be further elucidated through the characterization of these non-binding mutants in cells.
Topics: Animals; Binding Sites; COS Cells; Chlorocebus aethiops; Class I Phosphatidylinositol 3-Kinases; Dogs; Immunoblotting; Immunoprecipitation; Phosphatidylinositol 3-Kinase; rab5 GTP-Binding Proteins
PubMed: 29170408
DOI: 10.1038/s41598-017-16029-6