Effects of oxidation agents and metal ions on binding of p53 to supercoiled DNA

Wild type human full length (f.l.) tumor suppressor p53 protein binds preferentially to supercoiled (sc) DNA in vitro both in the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on the agarose gel. Bands revealed by immunoblotting with antibody DO-1 corresponded to the ethidium stained retarded bands. The intensity and the number of bands of p53-scDNA complex were decreased by physiological concentrations of unchelated zinc ions. Nickel and cobalt ions inhibited binding of p53 to scDNA and to p53CON in linear DNA fragments less efficiently than zinc. Compared to the intrinsic zinc strongly bound to Cys 176, Cys 238, Cys 242 and His 179 in the p53 core domain, binding of additional Zn2+ to p53 was much weaker as shown by an easy removal of the latter ions by low concentrations of EDTA. Oxidation of the protein with diamide resulted in a decrease of the number of the retarded bands. Under the same conditions, no binding of oxidized p53 to p53CON in a linear DNA fragment was observed. In agreement with the literature oxidation of f.l. p53 with diamide was irreversible and was not reverted by an excess of DTT. We showed that in the presence of 0.1 mM zinc ions, oxidation of p53 became reversible. Other divalent cations tested (cadmium, cobalt, nickel) exhibited no such effect. We suggested that the irreversibility of p53 oxidation was due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain (after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein) accompanied by a change in the p53 conformation. Binding of C-terminal anti-p53 antibody also protected bacterially expressed protein against irreversible loss of activity due to diamide oxidation. Binding the human p53 core domain (segment 94-312) to scDNA greatly differed from that observed with the full-length p53. The core domain did not posses the ability to bind strongly to many sites in scDNA regardless of the presence or absence of p53CON suggesting involvement of some other domain (probably C-terminal) in binding of the full-length p53 to scDNA. Supershift experiments using antibodies against p53 N- or C-terminus suggested that in oxidized p53, scDNA binding through the C-terminus gained importance

Myocardial Diseases: Current Views on Etiopathogenesis, Diagnostic Modalities, and Therapeutic Options.

According to current European classification, cardiomyopathies are defined as myocardial disorders that cannot be explained by coronary artery disease or abnormal loading conditions including valvular and congenital diseases. Six specific morphological and functional phenotypes are distinguished: hypertrophic, dilated, restrictive, and arrhythmogenic right ventricular cardiomyopathy together with two unclassified subtypes: Tako-tsubo and left ventricular noncompaction cardiomyopathies. In almost all of these phenotypes, inherited forms may be found; moreover, in some of them—for example, in hypertrophic cardiomyopathy—a genetic origin is even a rule. Therefore, genetic counseling shall be an integral part of the state-of-the-art care of patients with heart muscle disorders. The affected individuals together with their relatives have to be informed about the genetic basis of their disease and the potential risk for other family members. A detailed cardiac evaluation, including ECG and echocardiography (in some cases also Holter ECG monitoring), of first-degree relatives is necessary with their further regular follow-up. If available, genetic testing shall be discussed with the patients as it may improve their management as well as enable effective preventive genetic testing in other relatives

Biophysical and electrochemical studies of protein-nucleic acid interactions

This review is devoted to biophysical and electrochemical methods used for studying protein-nucleic acid (NA) interactions. The importance of NA structure and protein-NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein-DNA and protein-RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein-NA interactions. Recent applications of electrochemical methods in studies of protein-NA interactions are discussed in detail

Non-Penetrance for Ocular Phenotype in Two Individuals Carrying Heterozygous Loss-of-Function ZEB1 Alleles

ZEB1 loss-of-function (LoF) alleles are known to cause a rare autosomal dominant disorder—posterior polymorphous corneal dystrophy type 3 (PPCD3). To date, 50 pathogenic LoF variants have been identified as disease-causing and familial studies have indicated that the PPCD3 phenotype is penetrant in approximately 95% of carriers. In this study, we interrogated in-house exomes (n = 3616) and genomes (n = 88) for the presence of putative heterozygous LoF variants in ZEB1. Next, we performed detailed phenotyping in a father and his son who carried a novel LoF c.1279C>T; p.(Glu427*) variant in ZEB1 (NM_030751.6) absent from the gnomAD v.2.1.1 dataset. Ocular examination of the two subjects did not show any abnormalities characteristic of PPCD3. GnomAD (n = 141,456 subjects) was also interrogated for LoF ZEB1 variants, notably 8 distinct heterozygous changes presumed to lead to ZEB1 haploinsufficiency, not reported to be associated with PPCD3, have been identified. The NM_030751.6 transcript has a pLI score ≥ 0.99, indicating extreme intolerance to haploinsufficiency. In conclusion, ZEB1 LoF variants are present in a general population at an extremely low frequency. As PPCD3 can be asymptomatic, the true penetrance of ZEB1 LoF variants remains currently unknown but is likely to be lower than estimated by the familial led approaches adopted to date

Photogeneration of Spin Quintet Triplet–Triplet Excitations in DNA-Assembled Pentacene Stacks

Singlet fission (SF), an exciton-doubling process observed in certain molecular semiconductors where two triplet excitons are generated from one singlet exciton, requires correctly tuned intermolecular coupling to allow separation of the two triplets to different molecular units. We explore this using DNA-encoded assembly of SF-capable pentacenes into discrete π-stacked constructs of defined size and geometry. Precise structural control is achieved via a combination of the DNA duplex formation between complementary single-stranded DNA and the local molecular geometry that directs the SF chromophores into a stable and predictable slip-stacked configuration, as confirmed by molecular dynamics (MD) modeling. Transient electron spin resonance spectroscopy revealed that within these DNA-assembled pentacene stacks, SF evolves via a bound triplet pair quintet state, which subsequently converts into free triplets. SF evolution via a long-lived quintet state sets specific requirements on intermolecular coupling, rendering the quintet spectrum and its zero-field-splitting parameters highly sensitive to intermolecular geometry. We have found that the experimental spectra and zero-field-splitting parameters are consistent with a slight systematic strain relative to the MD-optimized geometry. Thus, the transient electron spin resonance analysis is a powerful tool to test and refine the MD-derived structure models. DNA-encoded assembly of coupled semiconductor molecules allows controlled construction of electronically functional structures, but brings with it significant dynamic and polar disorders. Our findings here of efficient SF through quintet states demonstrate that these conditions still allow efficient and controlled semiconductor operation and point toward future opportunities for constructing functional optoelectronic systems

Advancing human induced pluripotent stem cell-derived blood-brain barrier models for studying immune cell interactions.

Human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC-derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)-like cells with good barrier properties and mature tight junctions. Importantly, EECM-BMEC-like cells exhibited constitutive cell surface expression of ICAM-1, ICAM-2, and E-selectin. Pro-inflammatory cytokine stimulation increased the cell surface expression of ICAM-1 and induced cell surface expression of P-selectin and VCAM-1. Co-culture of EECM-BMEC-like cells with hiPSC-derived smooth muscle-like cells or their conditioned medium further increased the induction of VCAM-1. Functional expression of endothelial ICAM-1 and VCAM-1 was confirmed by T-cell interaction with EECM-BMEC-like cells. Taken together, we introduce the first hiPSC-derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions

Pigmentary retinopathy can indicate the presence of pathogenic LAMP2 variants even in somatic mosaic carriers with no additional signs of Danon disease

PURPOSE: Danon disease (DD) is a rare X-linked disorder caused by pathogenic variants in LAMP2. DD primarily manifests as a severe cardiomyopathy. An early diagnosis is crucial for patient survival. The aim of the study was to determine the usefulness of ocular examination for identification of DD. METHODS: Detailed ocular examination in 10 patients with DD (3 males, 7 females) and a 45-year-old asymptomatic female somatic mosaic carrier of a LAMP2 disease-causing variant. RESULTS: All patients with manifest cardiomyopathy had pigmentary retinopathy with altered autofluorescence and diffuse visual field loss. Best corrected visual acuity (BCVA) was decreased (<0.63) in 8 (40%) out of 20 eyes. The severity of retinal pathology increased with age, resulting in marked cone-rod involvement overtime. Spectral-domain optical coherence tomography in younger patients revealed focal loss of photoreceptors, disruption and deposition at the retinal pigment epithelium/Bruch's membrane layer (corresponding to areas of marked increased autofluorescence), and hyperreflective foci in the outer nuclear layer. Cystoid macular oedema was seen in one eye. In the asymptomatic female with somatic mosaicism, the BCVA was 1.0 bilaterally. An abnormal autofluorescence pattern in the left eye was present; while full-field electroretinography was normal. CONCLUSIONS: Detailed ocular examination may represent a sensitive and quick screening tool for the identification of carriers of LAMP2 pathogenic variants, even in somatic mosaicism. Hence, further investigation should be undertaken in all patients with pigmentary retinal dystrophy as it may be a sign of a life-threatening disease

Smc5/6: a link between DNA repair and unidirectional replication?

Of the three structural maintenance of chromosome (SMC) complexes, two directly regulate chromosome dynamics. The third, Smc5/6, functions mainly in homologous recombination and in completing DNA replication. The literature suggests that Smc5/6 coordinates DNA repair, in part through post-translational modification of uncharacterized target proteins that can dictate their subcellular localization, and that Smc5/6 also functions to establish DNA-damage-dependent cohesion. A nucleolar-specific Smc5/6 function has been proposed because Smc5/6 yeast mutants display penetrant phenotypes of ribosomal DNA (rDNA) instability. rDNA repeats are replicated unidirectionally. Here, we propose that unidirectional replication, combined with global Smc5/6 functions, can explain the apparent rDNA specificity

The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

© 2008 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3β homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.Deutsche Forschungsgemeinschaf