Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.     

Mitochondrial Dysfunction in Pten Haplo-Insufficient Mice with Social Deficits and Repetitive Behavior: Interplay between Pten and p53

Etiology of aberrant social behavior consistently points to a strong polygenetic component involved in fundamental developmental pathways, with the potential of being enhanced by defects in bioenergetics. To this end, the occurrence of social deficits and mitochondrial outcomes were evaluated in conditional Pten (Phosphatase and tensin homolog) haplo-insufficient mice, in which only one allele was selectively knocked-out in neural tissues. Pten mutations have been linked to Alzheimer's disease and syndromic autism spectrum disorders, among others. By 4-6 weeks of age, Pten insufficiency resulted in the increase of several mitochondrial Complex activities (II-III, IV and V) not accompanied by increases in mitochondrial mass, consistent with an activation of the PI3K/Akt pathway, of which Pten is a negative modulator. At 8-13 weeks of age, Pten haplo-insufficient mice did not show significant behavioral abnormalities or changes in mitochondrial outcomes, but by 20-29 weeks, they displayed aberrant social behavior (social avoidance, failure to recognize familiar mouse, and repetitive self-grooming), macrocephaly, increased oxidative stress, decreased cytochrome c oxidase (CCO) activity (50%) and increased mtDNA deletions in cerebellum and hippocampus. Mitochondrial dysfunction was the result of a downregulation of p53-signaling pathway evaluated by lower protein expression of p21 (65% of controls) and the CCO chaperone SCO2 (47% of controls), two p53-downstream targets. This mechanism was confirmed in Pten-deficient striatal neurons and, HCT 116 cells with different p53 gene dosage. These results suggest a unique pathogenic mechanism of the Pten-p53 axis in mice with aberrant social behavior: loss of Pten (via p53) impairs mitochondrial function elicited by an early defective assembly of CCO and later enhanced by the accumulation of mtDNA deletions. Consistent with our results, (i) SCO2 deficiency and/or CCO activity defects have been reported in patients with learning disabilities including autism and (ii) mutated proteins in ASD have been found associated with p53-signaling pathways.     

Heme Oxygenase-1 Regulates the Progression of K/BxN Serum Transfer Arthritis

Background

Heme oxygenase-1 (HO-1) is induced in many cell types as a defense mechanism against stress. We have investigated the possible role of endogenous HO-1 in the effector phase of arthritis using the K/BxN serum transfer model of arthritis in HO-1 heterozygous and homozygous knock-out mice.

Methodology/Principal Findings

Arthritis was induced in C57/Black-6 xFVB (HO-1^+/+, HO-1^+/− and HO-1^−/−) mice by intraperitoneal injection of 150 µl serum from arthritic K/BxN mice at days 0 and 2. Blood was collected and animals were sacrificed at day 10. Histological analysis was performed in ankle sections. The levels of inflammatory mediators were measured in serum and paw homogenates by enzyme-linked immunosorbent assay or Multiplex technology. The incidence of arthritis was higher in HO-1^+/− and HO-1^−/− groups compared with HO-1^+/+. The inflammatory response was aggravated in HO-1^+/− mice as shown by arthritic score and the migration of inflammatory cells that could be related to the enhancement of CXCL-1 production. In addition, the HO-1^+/− group showed proteoglycan depletion significantly higher than HO-1^+/+ mice. Serum levels of matrix metalloproteinase-3, monocyte chemotactic protein-1, plasminogen activator inhibitor-1, E-selectin and intercellular adhesion molecule-1 were increased in arthritic HO-1^−/− mice, whereas vascular endothelial growth factor and some cytokines such as interferon-γ showed a reduction compared to HO-1^+/+ or HO-1^+/− mice. In addition, down-regulated gene expression of ferritin, glutathione S-reductase A1 and superoxide dismutase-2 was observed in the livers of arthritic HO-1^+/− animals.

Conclusion/Significance

Endogenous HO-1 regulates the production of systemic and local inflammatory mediators and plays a protective role in K/BxN serum transfer arthritis.     

Inhibition of dendritic cell maturation and function is independent of heme oxygenase 1 but requires the activation of STAT3

The induction of heme oxygenase 1 (HO-1) by a single treatment with cobalt protoporphyrin (CoPPIX) protects against inflammatory liver failure and ischemia reperfusion injury after allotransplantation. In this context, the HO-1-mediated inhibition of donor-derived dendritic cell maturation and migration is discussed as one of the key events of graft protection. To investigate the poorly understood mechanism of CoPPIX-induced HO-1 activity in more detail, we performed gene expression analysis in murine liver, revealing the up-regulation of STAT3 after CoPPIX treatment. By using wild-type and HO-1-deficient dendritic cells we demonstrated that LPS-induced maturation is dependent on STAT3 phosphorylation and independent of HO-1 activity. In summary, our observations revise our understanding of the anti-inflammatory properties of HO-1 and highlight the immunomodulatory capacity of STAT3, which might be of further interest for targeting undesired immune responses, including ischemia reperfusion injury.     

Amplification of a single-locus variable-number direct repeats with restriction fragment length polymorphism (DR-PCR/RFLP) for genetic typing of Acinetobacter baumannii strains

In search of an effective DNA typing technique for Acinetobacter baumannii strains for hospital epidemiology use, the performance and convenience of a new target sequence was evaluated. Using known genomic sequences of Acinetobacter baumannii strains AR 319754 and ATCC 17978, we developed single-locus variable-number direct-repeat analysis using polymerase chain reaction-restriction fragment length polymorphism (DR-PCR/RFLP) method. A total of 90 Acinetobacter baumannii strains isolated from patients of the Clinical Hospital in Bydgoszcz, Poland, were examined. Initially, all strains were typed using macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE). Digestion of the chromosomal DNA with the ApaI endonuclease and separation of the fragments by PFGE revealed 21 unique types. Application of DR-PCR/RFLP resulted in recognition of 12 clusters. The results showed that the DR-PCR/RFLP method is less discriminatory than REA-PFGE, however, the novel genotyping method can be used as an alternative technique for generating DNA profiles in epidemiological studies of intra-species genetic relatedness of Acinetobacter baumannii strains.     

Thiocyanate concentration in saliva of cystic fibrosis patients

Thiocyanates (SCN-) are ubiquitous in nature. There are indispensable part of host defense system that act as a substrate for lactoperoxidase (LPO). In our study we present initial data on SCN- concentration in saliva of CF patients in comparison to healthy non-smokers and healthy smokers. 5 ml of saliva was collected from each subject to a sterile tube and thiocyanate concentration was measured in each sample. The results of the measurements are presented on Fig. 1. Mean concentration of SCN- in saliva of CF patients was 0.031 +/- 0.0052 g/l, in healthy non-smokers 0.039 +/- 0.0048 g/l and in healthy smokers 0.048 +/- 0.0161 g/l. The differences between each group were statistically significant. Studies on larger group of patients and probably on different material (BALF or induced sputum) should present interesting data complementing the in vitro studies.     

Genes and structure of selected cytokines involved in pathogenesis of psoriasis

Psoriasis is a common skin disease involving 1-4% of human population worldwide, of strong genetic background. The following cytokines are directly involved in psoriasis: TNF, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-18, IL-19, IL-20, IL-23 whereas IL-4, IL-10, IL-12 as well as IL-11, IL-17 and IFN-gamma are rather indirectly engaged. This work is a review of some genetic factors and structure of selected cytokines and receptors and their genes location.     

Human cathepsin D

A literature survey was performed of human cathepsin D gene, cathepsin D biosynthesis, posttranslatory modifications, transport within the cell, substrate specificity and catalytic effect. Methods used to determine the activity and level of this proteinase as well as its role in the biochemistry and pathobiochemistry of cells, tissues and organs were considered.     

Aluminium-induced changes in the surface and micropore properties of wheat roots: a study using the water vapor adsorption-desorption technique

The geometric and energetic characteristics of root surfaces of two wheat (Triticum L.) varieties, Al tolerant (Inia 66/16) and Al sensitive (Henika), were estimated from experimental water vapor adsorption–desorption data. Roots stressed for around 1 week at pH 4 without and with a toxic aluminium level (0.741 mol m^–3) were studied at the tillering and shooting stages. Roots grown continuously at pH 7 were taken as control. The surface properties of the pH 4 stressed roots were apparently the same as those of the control roots whatever the root age. For the roots of both varieties, the surface area and total micropore volume increased markedly after aluminium treatment. The average micropore radius increased significantly for the sensitive wheat, whereas it increased only slightly for the resistant one. Under Al treatment the number of large pores increased while small pores were fewer for both plants, indicating a possible alteration of the build-up of root tissue. The root surface pores were fractal. The fractal dimension of the sensitive wheat roots decreased under Al treatment, whereas for the resistant wheat this remained apparently unchanged. The adsorption energy distribution functions had different shapes for the sensitive and the resistant wheat varieties: the sensitive variety had greater number of high energy adsorption centers, which implies that the root tolerance on Al stress may be connected with lower polarity of the surface.