Doxorubicin catalyses self-assembly of p53 by phase separation.

Liquid-liquid phase separation plays a crucial role in cellular physiology, as it leads to the formation of membrane-less organelles in response to various internal stimuli, contributing to various cellular functions. However, the influence of exogenous stimuli on this process in the context of disease intervention remains unexplored. In this current investigation, we explore the impact of doxorubicin on the abnormal self-assembly of p53 using a combination of biophysical and imaging techniques. Additionally, we shed light on the potential mechanisms behind chemoresistance in cancer cells carrying mutant p53. Our findings reveal that doxorubicin co-localizes with both wild-type p53 (WTp53) and its mutant variants. Our in vitro experiments indicate that doxorubicin interacts with the N-terminal-deleted form of WTp53 (WTp53ΔNterm), inducing liquid-liquid phase separation, ultimately leading to protein aggregation. Notably, the p53 variants at the R273 position exhibit a propensity for phase separation even in the absence of doxorubicin, highlighting the destabilizing effects of point mutations at this position. The strong interaction between doxorubicin and p53 variants, along with its localization within the protein condensates, provides a potential explanation for the development of chemotherapy resistance. Collectively, our cellular and in vitro studies emphasize the role of exogenous agents in driving phase separation-mediated p53 aggregation.

Doxorubicin induced aggregation of α-synuclein: Insights into the mechanism of drug induced Parkinsonism.

The aggregation of α-synuclein is a prominent feature of Parkinson's disease. It is induced by factors such as genetic mutations and presence of metal salts leading to Parkinson's like symptoms. Existing case studies show that patients undergoing cancer chemotherapeutics are also prone to developing Parkinson's like symptoms. However, the underlying cause behind onset of these symptoms is not understood. It is not clear whether the administration of chemotherapeutic drugs alter the structural stability of α-synuclein. In the present study, we address this question by looking into the effect of chemotherapeutic drug namely doxorubicin on the α-synuclein stability. Using complementary spectroscopic, molecular docking and imaging techniques, we observe that doxorubicin interacted with central aggregation prone region of α-synuclein and induces destabilization leading to aggregation. We also show that the combination of doxorubicin and L-DOPA drugs impedes the α-synuclein aggregation. This may explain the reason behind the effectiveness of using L-DOPA against Parkinson's like symptoms.

Variable Mutations at the p53-R273 Oncogenic Hotspot Position Leads to Altered Properties.

Mutations in p53 protein, especially in the DNA-binding domain, is one of the major hallmarks of cancer. The R273 position is a DNA-contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant variants of R273 in p53 have different survival rates, indicating that the DNA-contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the properties of three major oncogenic variants of the wild-type (WT) p53: [R273H]p53, [R273C]p53, and [R273L]p53. Using a series of biophysical, biochemical, and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but they also show distinct structural stability, aggregation, and toxicity profiles. The WTp53 and the [R273H]p53 show the least destabilization and aggregation propensity. [R273C]p53 aggregation is disulfide mediated, leading to cross-ß, thioflavin-T-positive aggregates, whereas hydrophobic interactions dominate self-assembly in [R273L]p53, leading to a mixture of amyloid and amorphous aggregates. Molecular dynamics simulations indicate different contact maps and secondary structures for the different variants along the course of the simulations. Our study indicates that each of the R273 variants has its own distinct property of stability and self-assembly, the molecular basis of which may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue-specific or process-specific protein aggregation as a target.

Comparison of three amyloid assembly inhibitors: the sugar scyllo-inositol, the polyphenol epigallocatechin gallate, and the molecular tweezer CLR01.

Many compounds have been tested as inhibitors or modulators of amyloid ß-protein (Aß) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimer's disease (AD). These compounds typically were either designed to break apart ß-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aß, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958-16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aß aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aß using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aß monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved.

A key role for lysine residues in amyloid ß-protein folding, assembly, and toxicity.

A combination of hydrophobic and electrostatic interactions is important in initiating the aberrant self-assembly process that leads to formation of toxic oligomers and aggregates by multiple disease-related proteins, including amyloid ß-protein (Aß), whose self-assembly is believed to initiate brain pathogenesis in Alzheimer's disease. Lys residues play key roles in this process and participate in both types of interaction. They also are the target of our recently reported molecular tweezer inhibitors. To obtain further insight into the role of the two Lys residues in Aß assembly and toxicity, here we substituted each by Ala in both Aß40 and Aß42 and studied the impact of the substitution on Aß oligomerization, aggregation, and toxicity. Our data show that each substitution has a major impact on Aß assembly and toxicity, with significant differences depending on peptide length (40 versus 42 amino acids) and the position of the substitution. In particular, Lys16→Ala substitution dramatically reduces Aß toxicity. The data support the use of compounds targeting Lys residues specifically as inhibitors of Aß toxicity and suggest that exploring the role of Lys residues in other disease-related amyloidogenic proteins may help understanding the mechanisms of aggregation and toxicity of these proteins.

Application of photochemical cross-linking to the study of oligomerization of amyloidogenic proteins.

Assembly of amyloidogenic proteins into toxic oligomers and fibrils is an important pathogenic feature of over 30 amyloid-related diseases. Understanding the structures and mechanisms involved in the assembly process is necessary for rational approaches geared at inhibiting formation of these toxic species. Here, we review the application of photo-induced cross-linking of unmodified proteins (PICUP) to two disease-related amyloidogenic proteins (1) islet amyloid polypeptide (IAPP), whose toxic oligomers are thought to cause the demise of pancreatic ß-cells in type-2 diabetes mellitus and (2) α-synuclein, which aggregates into toxic oligomers and precipitates in Lewy bodies in Parkinson's disease. PICUP is an effective method allowing chemical "freezing" of dynamically changing oligomers and subsequent study of the oligomer size distribution that existed before cross-linking. The method has provided insights into the factors controlling early oligomerization, which could not be obtained by other means. We discuss sample preparation, experimental details, optimization of parameters, and troubleshooting.

Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins.

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid ß-protein (Aß) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aß at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aß oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella.

Hundreds of bacterial species use microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of a protein shell encapsulating specific metabolic enzymes. In Salmonella, an MCP is used for 1,2-propanediol utilization (Pdu MCP). The shell of this MCP is composed of eight different types of polypeptides, but their specific functions are uncertain. Here, we individually deleted the eight genes encoding the shell proteins of the Pdu MCP. The effects of each mutation on 1,2-PD degradation and MCP structure were determined by electron microscopy and growth studies. Deletion of the pduBB', pduJ, or pduN gene severely impaired MCP formation, and the observed defects were consistent with roles as facet, edge, or vertex protein, respectively. Metabolite measurements showed that pduA, pduBB', pduJ, or pduN deletion mutants accumulated propionaldehyde to toxic levels during 1,2-PD catabolism, indicating that the integrity of the shell was disrupted. Deletion of the pduK, pduT, or pduU gene did not substantially affect MCP structure or propionaldehyde accumulation, suggesting they are nonessential to MCP formation. However, the pduU or pduT deletion mutants grew more slowly than the wild type on 1,2-PD at saturating B(12), indicating that they are needed for maximal activity of the 1,2-PD degradative enzymes encased within the MCP shell. Considering recent crystallography studies, this suggests that PduT and PduU may mediate the transport of enzyme substrates/cofactors across the MCP shell. Interestingly, a pduK deletion caused MCP aggregation, suggesting a role in the spatial organization of MCP within the cytoplasm or perhaps in segregation at cell division.

Water-extracted polysaccharides from Azadirachta indica leaves: Structural features, chemical modification and anti-bovine herpesvirus type 1 (BoHV-1) activity.

In this study, we have analyzed the pectic arabinogalactan isolated from Azadirachta indica and its chemically sulfated derivative. The native polysaccharide had an apparent molecular mass of 80 kDa and is made up of (1→5)-/(1→3,5)-linked α-L-arabinosyl, (1→3)-/(1→6)-/(1→3,6)-linked ß-D-galactosyl, and terminal-rhamnosyl and α-l-arabinosyl residues. These macromolecules showed activity against bovine herpesvirus type-1. Their inhibitory concentration 50% values ranging from 31.12 to 105.25 µg/ml were lower than the cytotoxicity values (>1600-1440 µg/ml). The anti-viral effect was exerted during virus adsorption to the cell. Anionic groups in particular the sulfate groups appeared to be very important for the anti-herpetic activity of these polymers.

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family.

N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.

Form and dimensions of aggregates dictate cytotoxicities of Danish dementia peptides.

Familial Danish dementia (FDD) is a neurodegenerative disease which results due to alterations in the BRI2 gene. The pathological symptoms of the disease are cerebral amyloidolysis, parenchymal protein deposits and neuronal degeneration. The ADan peptide is a 34 amino acid long peptide which is thought to be the major cause of amyloid deposition in brains of patients suffering from FDD. Due to the presence of two cysteine residues viz. cys5 and cys23, this peptide exists in two forms: a cyclic oxidized form where the two cysteines form a disulfide bridge and a linear reduced form where the sulphydryl groups of cysteine are free. The relationship between toxicity and structure of the reduced and oxidized forms of ADan peptides has been elucidated by a combination of biophysical and cellular toxicity assays. It is observed that the reduced peptide has a stronger lethal effect on neuronal cell lines compared to its oxidized counterparts at all stages of aggregation. Further, it is observed that the fresh reduced peptide induced greater cell death as compared to its aged counterpart.

Fibrillogenesis in ADan peptides is inhibited by biphenyl ethers.

In this study, biphenyl ethers of diverse functionality were used to assess their effect on fibrillogenesis of both the oxidized and reduced ADan peptides, in vitro. It was noted that these compounds not only stalled fibrillogenesis but were also able to disrupt pre-formed fibers. The EC(50) values for the inhibition of this process lie in the nanomolar range for 50 microM of peptide concentration, indicating the high potency of these compounds as inhibitors. It was found that these compounds impart to the peptides, an alpha-helical conformation which does not allow them to aggregate and form fibrils. These studies also point out that the transition of peptides through alpha-helical conformation may be a prelude to the onset of fibrillogenesis for oxADan peptides.

Deuterium oxide stabilizes conformation of tubulin:a biophysical and biochemical study.

The present study was aimed to elucidate the mechanism of stabilization of tubulin by deuterium oxide (D(2)O). Rate of decrease of tryptophan fluorescence during aging of tubulin at 4 degrees C and 37 degrees C was significantly lower in D(2)O than in H(2)O. Circular dichroism spectra of tubulin after incubation at 4 degrees C, suggested that complete stabilization of the secondary structure in D(2)O during the first 24 hours of incubation. The number of available cysteine measured by DTNB reaction was decreased to a lesser extent in D(2)O than in H(2)O. During the increase in temperature of tubulin, the rate of decrease of fluorescence at 335 nm and change of CD value at 222 nm was lesser in D(2)O. Differential Scanning calorimetric experiments showed that the T(m) values for tubulin unfolding in D(2)O were 58.6 degrees C and 62.17 degrees C, and in H(2)O those values were 55.4 degrees C and 59.35 degrees C.

Design of mechanism-based inhibitors of transthyretin amyloidosis: studies with biphenyl ethers and new structural templates.

Transthyretin (TTR), a tetrameric thyroxine (T4) carrier protein, is associated with a variety of amyloid diseases. In this study, we explore the potential of biphenyl ethers (BPE), which are shown to interact with a high affinity to its T4 binding site thereby preventing its aggregation and fibrillogenesis. They prevent fibrillogenesis by stabilizing the tetrameric ground state of transthyretin. Additionally, we identify two new structural templates (2-(5-mercapto-[1,3,4]oxadiazol-2-yl)-phenol and 2,3,6-trichloro-N-(4H-[1,2,4]triazol-3-yl) represented as compounds 11 and 12, respectively, throughout the manuscript) exhibiting the ability to arrest TTR amyloidosis. The dissociation constants for the binding of BPEs and compound 11 and 12 to TTR correlate with their efficacies of inhibiting amyloidosis. They also have the ability to inhibit the elongation of intermediate fibrils as well as show nearly complete (>90%) disruption of the preformed fibrils. The present study thus establishes biphenyl ethers and compounds 11 and 12 as very potent inhibitors of TTR fibrillization and inducible cytotoxicity.

Isothermal unfolding studies on the apo and holo forms of Plasmodium falciparum acyl carrier protein. Role of the 4'-phosphopantetheine group in the stability of the holo form of Plasmodium falciparum acyl carrier protein.

The unfolding pathways of the two forms of Plasmodium falciparum acyl carrier protein, the apo and holo forms, were determined by guanidine hydrochloride-induced denaturation. Both the apo form and the holo form displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provides values for the conformational stability of the two proteins. Although both forms have the same amino acid sequence, and they have similar secondary structures, it was found that the - DeltaG of unfolding of the holo form was lower than that of the apo form at all the temperatures at which the experiments were done. The higher stability of the holo form can be attributed to the number of favorable contacts that the 4'-phosphopantetheine group makes with the surface residues by virtue of a number of hydrogen bonds. Furthermore, there are several hydrophobic interactions with 4'-phosphopantetheine that firmly maintain the structure of the holo form. We show here for the first time that the interactions between 4'-phosphopantetheine and the polypeptide backbone of acyl carrier protein stabilize the protein. As Plasmodium acyl carrier protein has a similar secondary structure to the other acyl carrier proteins and acyl carrier protein-like domains, the detailed biophysical characterization of Plasmodium acyl carrier protein can serve as a prototype for the analysis of the conformational stability of other acyl carrier proteins.

Structural basis for the specificity of basic winged bean lectin for the Tn-antigen: a crystallographic, thermodynamic and modelling study.

The crystal structure of winged bean basic agglutinin in complex with GalNAc-alpha-O-Ser (Tn-antigen) has been elucidated at 2.35 angstroms resolution in order to characterize the mode of binding of Tn-antigen with the lectin. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the complexation of the lectin with Tn-antigen. Modeling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein.