scholarly journals A zinc-binding domain is required for targeting the maternal nuclear protein PwA33 to lampbrush chromosome loops.

1995 ◽  
Vol 131 (3) ◽  
pp. 563-570 ◽  
Author(s):  
M Bellini ◽  
J C Lacroix ◽  
J G Gall
Keyword(s):  
Metal Binding ◽  
Nuclear Protein ◽  
Tertiary Structure ◽  
Colorimetric Assay ◽  
Ring Finger ◽  
Germinal Vesicle ◽  
Site Directed Mutagenesis ◽  
Zinc Binding ◽  
Lampbrush Chromosomes

In oocytes of the newt Pleurodeles waltl, the maternal nuclear protein PwA33 occurs on the lampbrush chromosomes and in some nucleoplasmic particles of the germinal vesicle. PwA33 is a modular protein and we used site-directed mutagenesis to alter the sequences encoding two metal-binding regions, the C3HC4 (or RING finger) and B-box motifs. Several mutant clones were generated and their synthetic transcripts were injected into Pleurodeles oocytes for in vivo analysis. In the oocyte, all translation products localized in the germinal vesicle. Proteins encoded by RING finger mutant clones were distributed in a pattern identical to that of the wild type protein, but when His266 of the B-box was mutated, PwA33 failed to localize in the lampbrush chromosomes and the nucleoplasmic particles. Using an in vitro colorimetric assay, we demonstrated that PwA33 is a zinc-binding protein and that mutations in the RING finger and B-Box altered its metal-binding properties. The RING finger motif bound two Zn2+ ions and the binding ratios of several mutants were consistent with the tertiary structure recently proposed for this motif. The B-box coordinated one Zn2+ and this binding was inhibited by the His266 mutation. The failure of the His266 mutation to bind zinc and to localize properly within the germinal vesicle suggests that an intact B-box is required for normal functioning of the PwA33 protein in the oocyte.

scholarly journals ADEP1 activated ClpP1P2 macromolecule of Leptospira, an ideal Achilles’ heel to deregulate proteostasis and hamper the cell survival

2020 ◽  
Author(s):  
Anusua Dhara ◽  
Md Saddam Hussain ◽  
Shankar Prasad Kanaujia ◽  
Manish Kumar
Keyword(s):  
Tertiary Structure ◽  
Bacterial Species ◽  
Small Extent ◽  
The Self ◽  
Site Directed Mutagenesis ◽  
Peptidase Activity ◽  
Serine Residue ◽  
Protein Substrates ◽  
Unfolded Protein

ABSTRACTThe caseinolytic protease (ClpP) complex in Leptospira interrogans is unusual in its functional activation. The genus Leptospira has two ClpPs, ClpP1 and ClpP2, which transcribes independently, regardless it couples to form the active tetradecamer. Acyldepsipeptide (ADEP) antibiotic hampers the growth of numerous bacterial species by activating the target protein ClpP and dysregulating the physiological proteostasis within the cell. In vitro culture of the L. interrogans fortified with the ADEP impeded the spirochete growth accompanied by a more elongated morphology. The chemoactivation of the ClpP is conditional on the duration of the self-compartmentalization of each of the ClpP isoforms. The small extent (10 min) self-assembled ClpP1P2 revealed inhibition in the peptidase activity (7-fold) in the presence of the ADEP due to the self-cleavage of the ClpP subunits. On supplementation of the β-casein or bovine serum albumin, the peptidase activity of the ClpP1P2 (short-incubated) got enhanced by the ADEP, while the ClpP1P2 (long-incubated) activity was retained to the same level. ADEP can also switch on the ClpP1P2 from a strict peptidase into proteolytic machinery that discerns and degrades the unfolded protein substrates autonomous of the cognate chaperone ClpX. In consensus to the most prokaryotes with the multi ClpP variants, the computational prototype of the ClpP1P2 tertiary structure infers that the hydrophobic pocket wherein the ADEPs predominantly docks are present in the ClpP2 heptamer. Additionally, the dynamic light scattering and the site-directed mutagenesis of a catalytic serine residue in either of the ClpP isoforms proposes a second interaction site for the ADEP.

scholarly journals Zinc Binding Properties of Engineered RING Finger Domain of Arkadia E3 Ubiquitin Ligase

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Christos T. Chasapis ◽  
Ariadni K. Loutsidou ◽  
Malvina G. Orkoula ◽  
Georgios A. Spyroulias
Keyword(s):  
Ubiquitin Ligase ◽  
Structural Integrity ◽  
Recombinant Expression ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Ring Domain ◽  
Site Directed Mutagenesis ◽  
Zinc Binding ◽  
Ring Finger Domain ◽  
C Terminus

Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF- signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues or to Arginine, through site-directed mutagenesis. The recombinant expression, inEscherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct.

Nonhistone Proteins of the Oocyte Nucleus of the Newt

1974 ◽  
Vol 15 (1) ◽  
pp. 145-161
Author(s):  
R. J. HILL ◽  
K. MAUNDRELL ◽  
H. G. CALLAN
Keyword(s):  
Nuclear Membrane ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Germinal Vesicle ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Nonhistone Proteins ◽  
Aerial Oxidation ◽  
A Minor

Evidence has been obtained which indicates that disulphide bond crosslinks contribute to the morphological integrity of isolated lampbrush chromosomes (both chromomeres and lateral loops) and nucleoli. It is suggested that the progressive formation of these bonds in vitro by aerial oxidation may provide the basis for the previously recognized time-dependent hardening or ‘denaturing’ of these structures. Manually isolated germinal vesicle nuclei have been massed and fractionated by low-speed centrifugation into nucleoplasm and chromatin. Phase-contrast microscopy demonstrates the chromatin to consist of nucleoli, lampbrush chromosomes and nuclear membranes. Urea gel electrophoresis has been employed to resolve the reduced and S-carboxymethylated proteins of whole nuclei into some 12 components, negatively charged at pH 8. The nucleoplasm alone gives an essentially similar pattern, but with the distinct depletion of one component and slight depletion of another. Both of these components are much enriched in the chromatin pellet where they predominate over all other proteins. The total chromatin has been subfractionated by microdissection, taking advantage of the differential attachment of nucleoli to the nuclear membrane at different stages of oogenesis. It is concluded that the nuclear membrane per se does not contribute to the major chromatin proteins. The two major polypeptides are components of the nucleoli. Preparations of isolated lampbrush chromosomes have not, to date, provided sufficient material to give a distinctive electropherogram; only one faint band, a major component of whole nuclei, was apparent. Sodium dodecyl sulphate gel electrophoresis has resolved some 25 components in whole nuclei, and again demonstrates the enrichment of the two major species in the total chromatin fraction. The apparent molecular weights of these two species are 43 kilodaltons and 110 kilodaltons. Approximately 20 minor species are also present in the chromatin and are obviously good candidates as components of the nucleolar and chromosomal structures. Histones, at most, make only a minor contribution to the overall chromatin protein population.

scholarly journals Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

2020 ◽  
Vol 295 (18) ◽  
pp. 6177-6186 ◽  
Author(s):  
Dongyang Huang ◽  
Sai Shi ◽  
Ce Liang ◽  
Xiaoyu Zhang ◽  
Xiaona Du ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Binding Site ◽  
Metal Binding ◽  
Recombinant Protein Expression ◽  
Rhythmic Activity ◽  
Oxidative Modification ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Zinc Binding ◽  
Patch Clamp Recording

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Future prospects for genetic manipulation of Rubisco

1986 ◽  
Vol 313 (1162) ◽  
pp. 459-469 ◽  
Keyword(s):  
Tertiary Structure ◽  
Genetic Manipulation ◽  
Chloroplast Transformation ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Lack Of Information ◽  
Eukaryotic Species ◽  
The Creation ◽  
Cloned Gene

Recent advances in the development of techniques for the manipulation of gene structure in vitro and genetic transformation of plants have brought the goal of directed genetic modification of RuBP carboxylase-oxygenase (Rubisco) within grasp. Genes from both prokaryotic and eukaryotic species have been cloned, sequenced and expressed in Escherichia coli , and in several instances this has resulted in the production of large quantities of fully functional enzyme. Several specifically-modified enzymes have been produced by site-directed mutagenesis of a cloned gene and the effects of the mutations evaluated following expression of the modified genes in E. coli . Thus, there are no major technical barriers to the creation and analysis of modified enzymes. A number of new opportunities now exist to explore the structural basis of naturally occurring differences in kinetic constants of the enzymes from diverse taxonomic sources. The recent report of chloroplast transformation mediated by the Ti plasmid has also raised the possibility that, if useful natural variation can be identified, genes for both the large and small subunits of the enzyme may eventually be transferred between species. However, the opportunities for rational application of mutagenesis in vitro in the creation of useful or informative variants of the enzyme is currently limited by lack of information about tertiary structure and the role of specific residues in catalysis.

scholarly journals The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence

2015 ◽  
Vol 83 (7) ◽  
pp. 2944-2956 ◽  
Author(s):  
Kathryn P. Haley ◽  
Alberto G. Delgado ◽  
M. Blanca Piazuelo ◽  
Brittany L. Mortensen ◽  
Pelayo Correa ◽  
...  
Keyword(s):  
Metal Binding ◽  
Binding Activity ◽  
S100 Proteins ◽  
Zinc Binding ◽  
Antimicrobial Protein ◽  
Secretion Systems ◽  
Content Type ◽  
S100a12 Protein ◽  
H Pylori

During infectious processes, antimicrobial proteins are produced by both epithelial cells and innate immune cells. Some of these antimicrobial molecules function by targeting transition metals and sequestering these metals in a process referred to as “nutritional immunity.” This chelation strategy ultimately starves invading pathogens, limiting their growth within the vertebrate host. Recent evidence suggests that these metal-binding antimicrobial molecules have the capacity to affect bacterial virulence, including toxin secretion systems. Our previous work showed that the S100A8/S100A9 heterodimer (calprotectin, or calgranulin A/B) binds zinc and represses the elaboration of theH. pyloricagtype IV secretion system (T4SS). However, there are several other S100 proteins that are produced in response to infection. We hypothesized that the zinc-binding protein S100A12 (calgranulin C) is induced in response toH. pyloriinfection and also plays a role in controllingH. pylorigrowth and virulence. To test this, we analyzed gastric biopsy specimens fromH. pylori-positive and -negative patients for S100A12 expression. These assays showed that S100A12 is induced in response toH. pyloriinfection and inhibits bacterial growth and viabilityin vitroby binding nutrient zinc. Furthermore, the data establish that the zinc-binding activity of the S100A12 protein represses the activity of thecagT4SS, as evidenced by the gastric cell “hummingbird” phenotype, interleukin 8 (IL-8) secretion, and CagA translocation assays. In addition, high-resolution field emission gun scanning electron microscopy (FEG-SEM) was used to demonstrate that S100A12 represses biogenesis of thecagT4SS. Together with our previous work, these data reveal that multiple S100 proteins can repress the elaboration of an oncogenic bacterial surface organelle.

Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries

2002 ◽  
Vol 30 (6) ◽  
pp. 1145-1149 ◽  
Author(s):  
Y. Takagi ◽  
E. Suyama ◽  
H. Kawasaki ◽  
M. Miyagishi ◽  
K. Taira
Keyword(s):  
Metal Binding ◽  
Cancer Metastasis ◽  
Tertiary Structure ◽  
Hammerhead Ribozyme ◽  
Rapid Identification ◽  
Specific Sequence ◽  
Target Sites ◽  
Interfering Rna

A hammerhead ribozyme was demonstrated to be a metalloenzyme. By controlling the metal-binding ability of the hammerhead ribozyme in the presence or absence of a specific sequence of interest, we engineered an allosterically controllable ribozyme, designated the maxizyme. Hybrid ribozymes were then constructed by coupling the site-specific cleavage activity of a hammerhead ribozyme with the unwinding activity of an endogenous RNA helicase. This leads to extremely efficient cleavage of target mRNA, not only in vitro, but also in vivo, and eliminates one of the major problems arising in the application of ribozymes for cleavage of mRNA in vivo: that many target sites on the RNA were previously inaccessible to cleavage owing to secondary and/or tertiary structure formation. Since hybrid ribozymes can efficiently attack target sites within mRNA, libraries were made of hybrid ribozymes with randomized binding arms, which were then introduced into cells. This procedure made it possible to readily identify the relevant genes associated with a specific phenotype, such as in apoptosis and cancer metastasis pathways. This application of a randomized library of hybrid ribozymes represents a simple, yet powerful, method for the identification of genes associated with specific phenotypes in the post-genome era. Moreover, vector-based siRNA (short-interfering RNA for RNA interference, RNAi) can also be used for the creation of the libraries and for the subsequent confirmation of the identified genes, relevant in the examined phenotype.

scholarly journals Mutation of the Zinc-Binding Metalloprotease Motif Affects Bacteroides fragilis Toxin Activity but Does Not Affect Propeptide Processing

2005 ◽  
Vol 73 (8) ◽  
pp. 5273-5277 ◽  
Author(s):  
Augusto A. Franco ◽  
Simy L. Buckwold ◽  
Jai W. Shin ◽  
Miguel Ascon ◽  
Cynthia L. Sears
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Bacteroides Fragilis ◽  
Site Directed Mutagenesis ◽  
Zinc Binding ◽  
Consensus Sequences ◽  
Biologic Activity ◽  
Single Point Mutations

ABSTRACT To evaluate the role of the zinc-binding metalloprotease in Bacteroides fragilis toxin (BFT) processing and activity, the zinc-binding consensus sequences (H348, E349, H352, G355, H358, and M366) were mutated by site-directed-mutagenesis. Our results indicated that single point mutations in the zinc-binding metalloprotease motif do not affect BFT processing but do reduce or eliminate BFT biologic activity in vitro.

scholarly journals Purification and properties of rat cysteine-rich intestinal protein

1994 ◽  
Vol 299 (2) ◽  
pp. 445-450 ◽  
Author(s):  
C Khoo ◽  
R J Cousins
Keyword(s):  
Small Intestine ◽  
Binding Affinity ◽  
Zinc Finger ◽  
Metal Binding ◽  
Tissue Concentration ◽  
Exchange Chromatography ◽  
Zinc Binding ◽  
Intestinal Lumen ◽  
Intestinal Protein

Cysteine-rich intestinal protein (CRIP) is a zinc-binding protein where the binding domain is in the so-called LIM double zinc finger motif. Methods are described for the preparation of CRIP from rat small intestine. Gel-filtration and ion-exchange chromatography and preparative PAGE gave homogeneous CRIP, based upon analytical PAGE, mass spectrometry and microsequencing. Initial localization of CRIP during chromatography was based on binding of 65Zn radioisotope introduced into the intestine. The stoichiometry of binding by CRIP is less than 2 atoms of zinc per molecule. The metal-binding affinity in vitro is zinc > cadmium > copper > iron, at low metal concentrations. Zinc is the predominant metal bound when these metals are taken up from the intestinal lumen. Zinc binding was not influenced by pH between values of 4.5 to 7.5. Metallothionein has a much greater zinc-binding affinity than CRIP. The tissue concentration of CRIP is of the order of 15-20 micrograms/g of mucosal tissue, suggesting that the protein is more abundant than zinc-finger-containing transcription factors. The metal-binding properties of CRIP are consistent with proposed zinc-related functions for this cytoplasmic protein, which is expressed in the small intestine during the postnatal period.

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