scholarly journals Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Taylor R. Cole ◽  
Tatyana I. Igumenova
Keyword(s):  
Lipid Membranes ◽  
Metal Ion ◽  
Membrane Binding ◽  
Regulatory Region ◽  
Signaling Proteins ◽  
Vis Spectroscopy ◽  
C1 Domain ◽  
Host Signaling ◽  
Coordination Spheres ◽  
And Function

Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd2+ as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity. Using solution NMR and UV-vis spectroscopy, we found that Cd2+ spontaneously replaced Zn2+ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd2+ substitution for Zn2+ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys3His metal-ion sites of C1B have higher affinity to Cd2+ than Zn2+, but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd2+. Our results indicate that Cd2+ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.

scholarly journals The Drosophila engrailed and invected Genes: Partners in Regulation, Expression and Function

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 893-906 ◽  
Author(s):  
Elizabeth Gustavson ◽  
Andrew S Goldsborough ◽  
Zehra Ali ◽  
Thomas B Kornberg
Keyword(s):  
Expression Pattern ◽  
Cultured Cells ◽  
Regulatory Region ◽  
Embryonic Lethality ◽  
Wild Type ◽  
Terminal Portion ◽  
Coding Sequence ◽  
Differential Effects ◽  
And Function ◽  
Redundant Functions

Abstract We isolated and characterized numerous engrailed and invected alleles. Among the deficiencies we isolated, a mutant lacking invected sequences was viable and phenotypically normal, a mutant lacking engrailed was an embryo lethal and had slight segmentation defects, and a mutant lacking both engrailed and invected was most severely affected. In seven engrailed alleles, mutations caused translation to terminate prematurely in the central or C-terminal portion of the coding sequence, resulting in embryonic lethality and segmentation defects. Both engrailed and invected expression declined prematurely in these mutant embryos. In wild-type embryos, engrailed and invected are juxtaposed and are expressed in essentially identical patterns. A breakpoint mutant that separates the mgrailed and invected transcription units parceled different aspects of the expression pattern to engrailed or invected. We also found that both genes cause similar defects when expressed ectopically and that the protein products of both genes act to repress transcription in cultured cells. We propose that the varied phenotypes of the engrailed alleles can be explained by the differential effects these mutants have on the combination of engrailed and invected activities, that engrailed and invected share a regulatory region, and that they encode redundant functions.

scholarly journals Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 222
Author(s):  
Agnieszka Polit ◽  
Paweł Mystek ◽  
Ewa Błasiak
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Lipid Membranes ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Membrane Attachment ◽  
The Individual ◽  
Multicellular Organisms ◽  
G Protein Coupled

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

scholarly journals Zn(II) and Ag(I) complexes of N-allythioamides of pyrimidinyl (cyclohexenyl) carboxylic acids and products their proton- and iodocyclization

2019 ◽  
Vol 85 (3) ◽  
pp. 3-19
Author(s):  
Polina Borovyk ◽  
Mariia Litvinchuk ◽  
Anton Bentya ◽  
Svitlana Orysyk ◽  
Yurii Zborovskiy ◽  
...  
Keyword(s):  
Carboxylic Acids ◽  
Metal Ion ◽  
Polymer Chains ◽  
Hydroxyl Group ◽  
Hydroxyl Groups ◽  
Polymer Complexes ◽  
Vis Spectroscopy ◽  
Polynuclear Coordination Compounds ◽  
Complexation Reactions ◽  
Oxygen Atoms

The possibility of using N-allylcarbothioamide derivatives as well as products of their iodine- and proton-initiated electrophilic heterocyclizations as chelating agents in complexation reactions with Zn(II) and Ag(I) ions is shown. Processing of the obtained experimental data showed that N-allythioamides of pyrimidinyl (cyclohexenyl) carboxylic acids H2L1 – H2L3 and their proton- and iodo-cyclization products HL4, HL5 containing four nucleophilic reaction centers (two oxygen atoms of the carbonyl and hydroxyl groups and N-, S-carbothioamide groups or N-atoms of the dihydrothiazole moiety) are polydentate ligands capable of coordinating with metal ions to form stable six-membered chelate metallocycles. A series of new chelating mono-, bi- and polynuclear complexes Zn(II) and Ag (I) of the composition [Zn2L1,32]n, [Zn2(HL1-3)2(CH3COO)2], [Ag2(HL1,3)2]n, [Zn(HL1-3)2], [Ag(H2L3)2NO3], [Zn(HL4,5)2], K[Ag(HL4,5)2] were synthesized and isolated in solid state. Their molecular structure was established by methods of elemental chemical analysis, NMR 1H, IR and UV-Vis spectroscopy. At a ratio of M:L 1:2, complexes were isolated in which two ligand molecules H2L1 − H2L3 are coordinated to the metal ion by the sulfur atoms of the carbothioamide group and the oxygen of the mono-deprotonated hydroxyl group. It was established that the products of the proton-/iodocyclization HL4, HL5 in the complex formation pass into the thione tautomeric form with coordination through the oxygen atoms of the deprotonated hydroxyl group and nitrogen atoms of the dihydrothiazole heterocycle. At M:L 1:1, binuclear or polynuclear coordination compounds are formed. It was shown that polymerisation in complexes [Zn2L1,32]n and [Ag2(HL1,3)2]n is due to the formation of Zn−(O2SN)−Zn and Ag−O−Ag polymer chains. Investigation of the solubility of the resulting complexes showed that the polymer complexes are weakly soluble or insoluble in DMSO, DMF, while the mononuclear are soluble in methanol, as well as in water.

scholarly journals Progressive methylation of POU5F1 regulatory regions during blastocyst development

Reproduction ◽  
2018 ◽  
Vol 156 (2) ◽  
pp. 145-161 ◽  
Author(s):  
E Canon ◽  
L Jouneau ◽  
T Blachère ◽  
N Peynot ◽  
N Daniel ◽  
...  
Keyword(s):  
De Novo ◽  
Regulatory Region ◽  
Upstream Region ◽  
Distal Enhancer ◽  
Blastocyst Development ◽  
Differentiated Cells ◽  
Rabbit Blastocyst ◽  
Highly Correlated ◽  
And Function

ThePOU5F1gene encodes one of the ‘core’ transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5′ regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere withPOU5F1expression and function inin vitro-derived cell lines. Also, its permanent inactivation in differentiated cells depends onde novomethylation of its promoter. However, little is known about the epigenetic regulation ofPOU5F1expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of thePOU5F15′ upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5′ regulatory region and the first exon accompanying differentiation and the gradual repression ofPOU5F1. Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.

PKCϵ has an alcohol-binding site in its second cysteine-rich regulatory domain

2009 ◽  
Vol 421 (3) ◽  
pp. 405-413 ◽  
Author(s):  
Joydip Das ◽  
Satyabrata Pany ◽  
Ghazi M. Rahman ◽  
Simon J. Slater
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
C1 Domain ◽  
Pkc Protein Kinase C ◽  
And Function

Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that an alcohol-binding site is present in PKCα in its C1 domain, which consists of two cysteine-rich subdomains, C1A and C1B. A PKCϵ-knockout mouse showed a significant decrease in alcohol consumption compared with the wild-type. The aim of the present study was to investigate whether an alcohol-binding site could be present in PKCϵ. Here we show that ethanol inhibited PKCϵ activity in a concentration-dependent manner with an EC50 (equilibrium ligand concentration at half-maximum effect) of 43 mM. Ethanol, butanol and octanol increased the binding affinity of a fluorescent phorbol ester SAPD (sapintoxin-D) to PKCϵC1B in a concentration-dependent manner with EC50 values of 78 mM, 8 mM and 340 μM respectively, suggesting the presence of an allosteric alcohol-binding site in this subdomain. To identify this site, PKCϵC1B was photolabelled with 3-azibutanol and 3-azioctanol and analysed by MS. Whereas azibutanol preferentially labelled His236, Tyr238 was the preferred site for azioctanol. Inspection of the model structure of PKCϵC1B reveals that these residues are 3.46 Å (1 Å=0.1 nm) apart from each other and form a groove where His236 is surface-exposed and Tyr238 is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding in terms of both photolabelling and alcohol-induced SAPD binding in the mutant H236A/Y238A. Whereas Tyr238 was labelled in mutant H236A, His236 was labelled in mutant Y238A. The present results provide direct evidence for the presence of an allosteric alcohol-binding site on protein kinase Cϵ and underscore the role of His236 and Tyr238 residues in alcohol binding.

scholarly journals A variant thrombasthenic phenotype associated with compound heterozygosity of integrin β3-subunit: (Met124Val)β3 alters the subunit dimerization rendering a decreased number of constitutive active αIIbβ3 receptors

2004 ◽  
Vol 92 (12) ◽  
pp. 1377-1386 ◽  
Author(s):  
Consuelo González-Manchón ◽  
Nora Butta ◽  
Susana Larrucea ◽  
Elena Arias-Salgado ◽  
Sonia Alonso ◽  
...  
Keyword(s):  
Metal Ion ◽  
Human Platelet ◽  
Cell Aggregation ◽  
Surface Expression ◽  
Paternal Allele ◽  
Constitutive Activity ◽  
Platelet Antigen ◽  
Human Platelet Antigen ◽  
And Function ◽  
Positively Charged

SummaryWe report the analysis of a variant case of thrombasthenic phenotype that is a compound heterozygote for two mutations located within the metal ion dependent adhesion site (MIDAS) of the β3 subunit.The patient inherited a maternal allele carrying the Met124Val substitution and a paternal allele that changes Asp119 to Tyr. Phenotyping of the human platelet antigen 1 (HPA-1) showed that the platelet αIIbβ3 complex in the patient was mostly accounted for by the Asp 119Tyr allele that does not bind to fibrinogen (Fg). The patient showed agonistinduced binding of platelets to Fg but neither binding to PAC-1 nor cell aggregation could be detected, most likely due to the minute expression (≤5%) of αIIb(124Val)β3 receptors. CHO cells expressing (124Val)β3 showed a diminished surface expression of αIIbβ3, enhanced adhesion to immobilized Fg, and spontaneous aggregation in the presence of soluble Fg, suggesting that (124Val)β3 may confer constitutive activity to the αIIb(124Val)β3 receptors. A distinct feature of these cells is the failure of DTT to enhance the binding to soluble Fg and the formation of cell aggregates. The substitution of (124Met)β3 by either a polar or a positively charged amino acid restored the surface exposure and function of the αIIbβ3 receptors whereas a negatively charged residue did not.

A FVIII C1 Domain Loop Containing Phe2093 Contributes Affinity to the Binding of Recombinant FVIII C1C2 Proteins to Activated Platelets

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3069-3069
Author(s):  
Ting-Chang Hsu ◽  
Kathleen P. Pratt ◽  
Arthur R. Thompson
Keyword(s):  
Membrane Binding ◽  
Cysteine Residue ◽  
Wild Type ◽  
Platelet Surface ◽  
Proteolytic Activation ◽  
Mutant Proteins ◽  
Activated Platelets ◽  
C1 Domain ◽  
Beta Hairpin ◽  
The Difference

Abstract Factor VIII (FVIII) circulates bound to von Willebrand factor, and upon proteolytic activation it dissociates and attaches to activated membranes, e.g. at the platelet surface, that expose negatively-charged phosphatidylserine. This membrane association is mediated entirely by the FVIIIa light chain (A3-C1-C2), and the C2 domain is known to be a primary contributor to the membrane affinity. We previously demonstrated that the C1 domain, which is homologous to C2, also contributes to the affinity for activated platelets, (Hsu et al., Blood111, 200–208, 2008). Our earlier work showed that the platelet affinity of recombinant C1C2, as well as the total number of binding sites per platelet, were significantly higher than those measured for recombinant C2. Thus C1 either interacts with the platelet surface directly, or it stabilizes a conformation of C2 that promotes membrane binding. In this study, the affinities for activated platelets of a series of mutant C1C2 proteins were evaluated to determine residues involved in FVIII binding to platelets. C1C2 and C2 proteins were generated with a free cysteine residue substituted for the wild-type serine at position 2296 in C2 (S2296C does not disrupt the protein structure or affect membrane binding), to which a sulfyhydryl-linked fluorescein probe was attached covalently (C1C2* and C2*). Single or double alanine substitutions were introduced at the two beta-hairpin turns in C2 that mediate membrane binding (M2199A, F2200A, L2251A, L2252A), and at C1 residue F2093, which aligns with C2 residue L2252. Washed platelets were activated with SFLLRN-amide, incubated for 1 hr with the wild-type or mutant proteins, and analyzed by flow cytometry on a FACSCaliber. The relative binding affinities were estimated by using multiple protein concentrations and comparing the fluorescent signals to the values for saturation binding of C1C2* and C2*. Alanine substitutions at all of these positions resulted in decreased binding of C1C2*, comparable to the difference in affinity of C2* versus C1C2* (figure 1A). C1C2-F2093C was then generated, and the introduced sulfhydryl was blocked by adding free cysteine, thus introducing the bulky, charged cystine residue at this putative membrane-binding site (C1C2-F2093C-C). Binding assays utilizing detection by monoclonal antibody ESH8, which does not interfere with membrane attachment, followed by a PE-labeled secondary antibody showed that C1C2-F2093C-C had markedly reduced binding to activated platelets compared to C1C2 (figure 1B). These results are consistent with the hypothesis that the C1 domain contacts the membrane directly when FVIIIa becomes attached to the activated platelet surface and indicate that F2093 contributes significantly to this interaction. Figure Figure

Sign in / Sign up

Export Citation Format

Share Document