scholarly journals Redox Sensitive Cysteine Residues as Crucial Regulators of Wild-Type and Mutant p53 Isoforms

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3149
Author(s):  
Elena Butturini ◽  
Giovanna Butera ◽  
Raffaella Pacchiana ◽  
Alessandra Carcereri de Prati ◽  
Sofia Mariotto ◽  
...  
Keyword(s):  
Redox State ◽  
P53 Protein ◽  
Tp53 Gene ◽  
Single Amino Acid ◽  
Cysteine Residues ◽  
Missense Mutations ◽  
Wild Type ◽  
Functional Changes ◽  
Type Protein ◽  
Wild Type Protein

The wild-type protein p53 plays a key role in preventing the formation of neoplasms by controlling cell growth. However, in more than a half of all cancers, the TP53 gene has missense mutations that appear during tumorigenesis. In most cases, the mutated gene encodes a full-length protein with the substitution of a single amino acid, resulting in structural and functional changes and acquiring an oncogenic role. This dual role of the wild-type protein and the mutated isoforms is also evident in the regulation of the redox state of the cell, with antioxidant and prooxidant functions, respectively. In this review, we introduce a new concept of the p53 protein by discussing its sensitivity to the cellular redox state. In particular, we focus on the discussion of structural and functional changes following post-translational modifications of redox-sensitive cysteine residues, which are also responsible for interacting with zinc ions for proper structural folding. We will also discuss therapeutic opportunities using small molecules targeting cysteines capable of modifying the structure and function of the p53 mutant isoforms in view of possible anticancer therapies for patients possessing the mutation in the TP53 gene.

Biochemical consequences of sedlin mutations that cause spondyloepiphyseal dysplasia tarda

2009 ◽  
Vol 423 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Mei Y. Choi ◽  
Caleb C. Y. Chan ◽  
Danny Chan ◽  
Keith D. K. Luk ◽  
Kathryn S. E. Cheah ◽  
...  
Keyword(s):  
Late Onset ◽  
Transport Protein ◽  
Missense Mutations ◽  
Wild Type ◽  
Spondyloepiphyseal Dysplasia ◽  
Type Protein ◽  
Wild Type Protein ◽  
Protein Particle ◽  
Spondyloepiphyseal Dysplasia Tarda

SEDT (spondyloepiphyseal dysplasia tarda) is a late-onset X-linked recessive skeletal dysplasia caused by mutations in the gene SEDL coding for sedlin. In the present paper, we investigated four missense mutations observed in SEDT and compare biochemical and cellular characteristics relative to the wild-type protein to address the mechanism of disease and to gain insight into the function of the sedlin protein. In situ hybridization and immunohistochemical experiments in mouse growth plates revealed sedlin to be predominantly expressed in proliferating and hypertrophic chondrocytes. Cell culture studies showed that the wild-type protein localized predominantly in the vicinity of the nucleus and the Golgi, with further localization around the cytoplasm, whereas mutation resulted in mislocalization. The D47Y mutant was expressed similarly to the wild-type, but the S73L, F83S and V130D mutants showed particularly low levels of expression that were rescued in the presence of the proteasome inhibitor MG132 (benzyloxycarbonyl-leucylleucylleucinal). Furthermore, whereas the D47Y mutant folded similarly and had similar stability to the wild-type sedlin as shown by CD and fluorescence, the S73L, F83S and V130D mutants all misfolded during expression. Two independent assays showed that the D47Y mutation resulted in an increased affinity for the transport protein particle component Bet3 compared with the wild-type sedlin. Our results suggest that the sedlin mutations S73L, F83S and V130D cause SEDT by sedlin misfolding, whereas the D47Y mutation may influence normal TRAPP (transport protein particle) dynamics.

scholarly journals The Human CC Chemokine MIP-1β Dimer Is Not Competent to Bind to the CCR5 Receptor

2007 ◽  
Vol 282 (38) ◽  
pp. 27976-27983 ◽  
Author(s):  
Hongjun Jin ◽  
Xiaohong Shen ◽  
Brandi Renee Baggett ◽  
Xiangming Kong ◽  
Patricia J. LiWang
Keyword(s):  
Analytical Ultracentrifugation ◽  
Single Amino Acid ◽  
Wild Type ◽  
Cc Chemokine ◽  
Definitive Evidence ◽  
Type Protein ◽  
Wild Type Protein ◽  
Inflammatory Protein ◽  
Ccr5 Receptor ◽  
Covalent Dimer

Chemokine dimerization has been the subject of much interest in recent years as evidence has accumulated that different quaternary states of chemokines play different biological roles; the monomer is believed to be the receptor-binding unit, whereas the dimer has been implicated in binding cell surface glycosaminoglycans. However, although several studies have provided evidence for this paradigm by making monomeric chemokine variants or dimer-impaired chemokines, few have provided direct evidence of the receptor function of a chemokine dimer. We have produced a covalent dimer of the CC chemokine macrophage inflammatory protein-1β (MIP-1β) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1β-A10C). This variant was shown to be a nondissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of glycosaminoglycan binding, MIP-1β-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1β-A10C neither binds nor activates the MIP-1β receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide cross-link by incubation with 1 mm dithiothreitol. This work provides the first definitive evidence that the CC chemokine MIP-1β dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.

scholarly journals Wild-type and missense mutants of retinoschisin co-assemble resulting in either intracellular retention or incorrect assembly of the functionally active octamer

2009 ◽  
Vol 425 (1) ◽  
pp. 275-284 ◽  
Author(s):  
Lindsay J. Gleghorn ◽  
Dorothy Trump ◽  
Neil J. Bulleid
Keyword(s):  
Missense Mutations ◽  
Heterogeneous Mixture ◽  
Wild Type ◽  
Functional Protein ◽  
Gene Encoding ◽  
Unfolded Protein ◽  
Type Protein ◽  
Wild Type Protein ◽  
Missense Mutant ◽  
Protein Response

The X-linked disease retinoschisis is caused by mutations in the RS1 gene encoding retinoschisin, most commonly missense mutations leading to a lack of secretion of functional protein. One potential approach to treat this disease would be the introduction of the wild-type protein by gene therapy in affected individuals. Retinoschisin normally forms homo-octamers, so co-expression of the wild-type protein with the mutant could result in their co-assembly. In the present study, we show that retinoschisin assembles into an octamer before transport from the endoplasmic reticulum and that co-assembly of wild-type and mutant protein can occur when they are co-expressed in the same cell. This co-assembly results in the retention of some, but not all, expressed wild-type retinoschisin. Moreover, when the wild-type protein is expressed with a missense mutant that is normally secreted, co-assembly occurs resulting in the secretion of a heterogeneous mixture of oligomers. Missense mutations of retinoschisin which cause intracellular retention also lead to an unfolded protein response. However, this is not sufficient to decrease cell viability suggesting that the pathology of the disease is not likely to be linked to programmed cell death.

Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA

2008 ◽  
Vol 414 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Allison Lewin ◽  
Allister Crow ◽  
Christopher T. C. Hodson ◽  
Lars Hederstedt ◽  
Nick E. Le Brun
Keyword(s):  
Active Site ◽  
Protein Unfolding ◽  
Cysteine Residues ◽  
Wild Type ◽  
Pka Values ◽  
Active Site Cysteine ◽  
Reduction Potentials ◽  
Type Protein ◽  
Wild Type Protein

The thiol–disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pKa values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in that they are both high (>8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid–base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pKa values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, ΔpKa values of −2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited an increase of +40 mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to the oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.

scholarly journals Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0128954 ◽  
Author(s):  
Saara Laulumaa ◽  
Tuomo Nieminen ◽  
Mari Lehtimäki ◽  
Shweta Aggarwal ◽  
Mikael Simons ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Neutron Scattering ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Peripheral Membrane Protein

Two novel mutations in Gli-similar 3 in patients with congenital hypothyroidism and thyroid dysgenesis

2021 ◽  
Author(s):  
Jie Lan ◽  
Chunhui Sun ◽  
Xinping Liang ◽  
Ruixin Ma ◽  
Yuhua Ji ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Transcriptional Activation ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Thyroid Dysgenesis ◽  
Type Protein ◽  
Wild Type Protein

Abstract Background: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). As variants of the transcription factor Gli-similar 3 (GLIS3) have been associated with CH and GLIS3 is one of candidate genes of TD, we screened and characterized GLIS3 mutations in Chinese patients with CH and TD.Methods: To detect mutations, we sequenced all GLIS3 exons in the peripheral blood genomic DNA isolated from 50 patients with TD and 100 healthy individuals. Wild-type and mutant expression vectors of Glis3 were constructed. Quantitative real-time PCR, western blotting, and double luciferase assay were performed to investigation the effect of the mutations on GLIS3 protein function and transcriptional activation.Results: Two novel heterozygous missense mutations, c.2710G>A (p.G904R) and c.2507C>A (p.P836Q), were detected in two unrelated patients. Functional studies revealed that p.G904R expression was 59.95% lower and p.P836Q was 31.23% lower than wild-type GLIS3 mRNA expression. The p.G904R mutation also resulted in lower GLIS3 protein expression compared with that encoded by wild-type GLIS3. Additionally, the luciferase reporter assay revealed that p.G904R mediated impaired transcriptional activation compared with the wild-type protein (p < 0.05) but did not have a dominant-negative effect on the wild-type protein.Conclusions: We for the first time screened and characterized the function of GLIS3 mutations in Chinese individuals with CH and TD. Our study not only broadens the GLIS3 mutation spectrum, but also provides further evidence that GLIS3 defects cause TD.

scholarly journals Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-Crystallin on Chaperone Function and Anti-Apoptotic Activity

2021 ◽  
Vol 22 (19) ◽  
pp. 10771
Author(s):  
Sundararajan Mahalingam ◽  
Srabani Karmakar ◽  
Puttur Santhoshkumar ◽  
Krishna K. Sharma
Keyword(s):  
Deletion Mutant ◽  
Mutant Protein ◽  
Wild Type ◽  
Chaperone Function ◽  
Type Protein ◽  
Wild Type Protein ◽  
Cytotoxicity Studies ◽  
Αb Crystallin ◽  
Apoptotic Activity ◽  
Subunit Organization

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

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