scholarly journals Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils

2021 ◽  
Vol 118 (10) ◽  
pp. e2015618118
Author(s):  
David S. Yang ◽  
Arash Saeedi ◽  
Aram Davtyan ◽  
Mohsen Fathi ◽  
Michael B. Sherman ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Amyloid Fibrils ◽  
Three Dimensional ◽  
Solution Concentration ◽  
Disordered Proteins ◽  
Mutant P53 ◽  
Multiple Length Scales ◽  
Activate Complex ◽  
Protein Rich ◽  
The Guardian

The protein p53 is a crucial tumor suppressor, often called “the guardian of the genome”; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

scholarly journals Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

2020 ◽  
Author(s):  
David S. Yang ◽  
Arash Saeedi ◽  
Aram Davtyan ◽  
Mohsen Fathi ◽  
Mohammad S. Safari ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Theoretical Models ◽  
Mutant P53 ◽  
Wild Type ◽  
Core Domain ◽  
Multiple Length Scales ◽  
Liquid Clusters ◽  
Activate Complex ◽  
Phase Behaviors ◽  
Protein Rich

AbstractThe oncogenic properties of mutant p53 have been ascribed to destabilization of the p53 conformation, followed by aggregation into insoluble fibrils. Here we combine immunofluorescent 3D confocal microscopy of breast cancer cells expressing the p53 mutant Arg248Gln (R248Q) with light scattering from solutions of the purified protein and molecular simulations to probe the mechanisms that govern phase behaviors of the mutant across multiple length scales, from cellular to molecular. We establish that p53 R248Q forms mesoscopic protein-rich clusters, an anomalous liquid phase with several unique properties. We demonstrate that the clusters host and facilitate the nucleation of amyloid fibrils. The distinct characteristics of the clusters of R248Q and wild-type p53 and theoretical models indicate that p53 condensation into clusters is driven by the structural destabilization of the core domain and not by interactions of its extensive disordered region. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster behaviors. In a broader context, our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

scholarly journals Suberoylanilide hydroxamic acid enhances the radiosensitivity of lung cancer cells through acetylated wild-type and mutant p53-dependent modulation of mitochondrial apoptosis

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098154
Author(s):  
Kan Wu ◽  
Xueqin Chen ◽  
Xufeng Chen ◽  
Shirong Zhang ◽  
Yasi Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Hydroxamic Acid ◽  
Site Directed Mutagenesis ◽  
Lung Cancer Cells ◽  
Mutant P53 ◽  
Suberoylanilide Hydroxamic Acid ◽  
Mitochondrial Apoptosis ◽  
Wild Type ◽  
Clonogenic Cell

Objective Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has shown potential as a candidate radiosensitizer for many types of cancers. This study aimed to explore the radiosensitization mechanism of SAHA in lung cancer cells. Methods Mutations in p53 were generated by site-directed mutagenesis using polymerase chain reaction. Transfection was performed to generate H1299 cells carrying wild-type or mutant p53. The radiosensitizing enhancement ratio was determined by clonogenic assays. Mitochondrial apoptosis was detected using JC-1 staining and flow cytometry analysis. Results Our results showed that SAHA induced radiosensitization in H1299 cells expressing wild-type p53, p53R175H or p53P223L, but this enhanced clonogenic cell death was not observed in parental H1299 (p53-null) cells or H1299 cells expressing p53 with K120R, A161T and V274R mutations. In SAHA-sensitized cells, mitochondrial apoptosis was induced following exposure to irradiation. Additionally, we observed that a secondary mutation at K120 (K120R) could eliminate p53-mediated radiosensitization and mitochondrial apoptosis. Conclusions The results of this study suggest that wild-type and specific mutant forms of p53 mediate SAHA-induced radiosensitization by regulating mitochondrial apoptosis, and the stabilization of K120 acetylation by SAHA is the molecular basis contributing to radiosensitization in lung cancer cells.

scholarly journals Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry

2020 ◽  
Author(s):  
Lasse Staby ◽  
Katherine R. Kemplen ◽  
Amelie Stein ◽  
Michael Ploug ◽  
Jane Clarke ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Three Dimensional ◽  
Life Time ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Order And Disorder ◽  
C Terminus ◽  
Close Relationship ◽  
Stability Dynamics ◽  
And Function

Abstract Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order–disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

Androgen receptor: acting in the three-dimensional chromatin landscape of prostate cancer cells

2011 ◽  
Vol 5 (1) ◽  
Author(s):  
Harri Makkonen ◽  
Jorma J. Palvimo
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Cancer Cells ◽  
Binding Sites ◽  
Target Genes ◽  
Three Dimensional ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Loop Formation ◽  
Prostate Cancer Cells

AbstractAndrogen receptor (AR) acts as a hormone-controlled transcription factor that conveys the messages of both natural and synthetic androgens to the level of genes and gene programs. Defective AR signaling leads to a wide array of androgen insensitivity disorders, and deregulated AR function, in particular overexpression of AR, is involved in the growth and progression of prostate cancer. Classic models of AR action view AR-binding sites as upstream regulatory elements in gene promoters or their proximity. However, recent wider genomic screens indicate that AR target genes are commonly activated through very distal chromatin-binding sites. This highlights the importance of long-range chromatin regulation of transcription by the AR, shifting the focus from the linear gene models to three-dimensional models of AR target genes and gene programs. The capability of AR to regulate promoters from long distances in the chromatin is particularly important when evaluating the role of AR in the regulation of genes in malignant prostate cells that frequently show striking genomic aberrations, especially gene fusions. Therefore, in addition to the mechanisms of DNA loop formation between the enhancer bound ARs and the transcription apparatus at the target core promoter, the mechanisms insulating distally bound ARs from promiscuously making contacts and activating other than their normal target gene promoters are critical for proper physiological regulation and thus currently under intense investigation. This review discusses the current knowledge about the AR action in the context of gene aberrations and the three-dimensional chromatin landscape of prostate cancer cells.

scholarly journals Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited

2009 ◽  
Vol 185 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Farideh Sabeh ◽  
Ryoko Shimizu-Hirota ◽  
Stephen J. Weiss
Keyword(s):  
Cancer Cells ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Amoeboid Movement ◽  
Type I ◽  
Collagen Network ◽  
Normal Tissues ◽  
Tissue Invasion ◽  
Absolute Requirement ◽  
Cross Links

Tissue invasion during metastasis requires cancer cells to negotiate a stromal environment dominated by cross-linked networks of type I collagen. Although cancer cells are known to use proteinases to sever collagen networks and thus ease their passage through these barriers, migration across extracellular matrices has also been reported to occur by protease-independent mechanisms, whereby cells squeeze through collagen-lined pores by adopting an ameboid phenotype. We investigate these alternate models of motility here and demonstrate that cancer cells have an absolute requirement for the membrane-anchored metalloproteinase MT1-MMP for invasion, and that protease-independent mechanisms of cell migration are only plausible when the collagen network is devoid of the covalent cross-links that characterize normal tissues.

Micropattern Effect on Breast Cancer Cells Behavior on Isotropically Etched Silicon Microenvironments

2010 ◽  
Author(s):  
Mehdi Nikkhah ◽  
Jeannine S. Strobl ◽  
Bhanu Peddi ◽  
Adedamola Omotosho ◽  
Masoud Agah
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Three Dimensional ◽  
Cancer Diagnostics ◽  
Breast Cancer Diagnostics ◽  
Wide Range ◽  
Etched Silicon ◽  
Directed Motility ◽  
Etched Surface ◽  
Silicon Based

In this paper we are investigating three dimensional (3-D) silicon-based microenvironments as potential platforms for breast cancer diagnostics. We have developed isotropically etched microstructures with a wide range of geometrical patterns for this purpose. Our results indicate that with the etched surface ratio of ∼65%, it is possible to capture 80–90% of the cancer cells within each silicon chip. After treatment of the cells with mitomycin C (to block the cell growth) more number of the cells are trapped inside the etched features for longer cultures times (72 h) suggesting that there is a directed motility and attraction of the cells toward the etched cavities and by optimally designing the etched features, the proposed platforms can be potentially used for diagnostics purposes.

Cationicity and hydrophobicity enhance the cytotoxic potency of Phoratoxin C anticancer peptide analogues against triple negative breast cancer cells

2021 ◽  
Vol 17 ◽  
Author(s):  
Chu Xin Ng ◽  
Cheng Foh Le ◽  
Sau Har Lee
Keyword(s):  
Cancer Cells ◽  
Anticancer Agents ◽  
Breast Cancer Cells ◽  
Critical Role ◽  
Three Dimensional ◽  
Vero Cells ◽  
Anticancer Peptides ◽  
Α Helix ◽  
Peptide Analogues

Background: Anticancer peptides (ACPs) have received increasing attention as a promising class of novel anticancer agents owing to its potent and rapid cytotoxic properties. In this study, we aim to investigate the effects of cationicity and hydrophobicity in modulating the cytotoxicity of PtxC, a class of ACP from the leafy mistletoe Phoradendron tomentosum against the MDA-MB-231 and Vero cells. Method: We designed a series of four PtxC analogues (PA1 – PA4) by residual substitutions with specific amino acids to introduce the specific charge and hydrophobicity alterations to the analogues. The cytotoxicity strength of the PtxC analogues on MDA-MB-231 and Vero cells were tested by using MTT assay at 24 hours post treatment. Results: PA1, PA2 and PA4 displayed marked increases in cytotoxicity against both MDA-MB-231 and Vero cells and can be ranked in the order of PA2 > PA4 > PA1 > PtxC > PA3. Sequence-activity relationship analyses of the designed analogues showed that an increase in the level of cationicity and hydrophobicity correlated well with the enhanced cytotoxic activity of PtxC analogues. This was observed with PA1 (netC +8) and PA2 (netC +10) in comparison to PtxC (netC +7). Similar finding was observed for PA4 (GRAVY +0.070) in contrast to PtxC (GRAVY -0.339). Three-dimensional modelling predicted a double α-helix structure in PtxC class of ACP. The larger first helix in PA2 and PA4 was suggested to be responsible for the enhanced cytotoxicity observed. Conclusion: The critical role of cationicity and hydrophobicity in enhancing cytotoxicity of PtxC class of ACPs were clearly demonstrated in our study. The current findings could be extrapolated to benefit peptide design strategy in other classes of ACPs toward the discovery of highly potent ACPs against cancer cells as potential novel therapeutic agents.

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