Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under 4 ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable supercomplex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.

Late-spring frost risk between 1959 and 2017 decreased in North America but increased in Europe and Asia.

Late-spring frosts (LSFs) affect the performance of plants and animals across the world's temperate and boreal zones, but despite their ecological and economic impact on agriculture and forestry, the geographic distribution and evolutionary impact of these frost events are poorly understood. Here, we analyze LSFs between 1959 and 2017 and the resistance strategies of Northern Hemisphere woody species to infer trees' adaptations for minimizing frost damage to their leaves and to forecast forest vulnerability under the ongoing changes in frost frequencies. Trait values on leaf-out and leaf-freezing resistance come from up to 1,500 temperate and boreal woody species cultivated in common gardens. We find that areas in which LSFs are common, such as eastern North America, harbor tree species with cautious (late-leafing) leaf-out strategies. Areas in which LSFs used to be unlikely, such as broad-leaved forests and shrublands in Europe and Asia, instead harbor opportunistic tree species (quickly reacting to warming air temperatures). LSFs in the latter regions are currently increasing, and given species' innate resistance strategies, we estimate that ∼35% of the European and ∼26% of the Asian temperate forest area, but only ∼10% of the North American, will experience increasing late-frost damage in the future. Our findings reveal region-specific changes in the spring-frost risk that can inform decision-making in land management, forestry, agriculture, and insurance policy.

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease.

The melibiose permease of Salmonella typhimurium (MelBSt) catalyzes the stoichiometric symport of galactopyranoside with a cation (H+, Li+, or Na+) and is a prototype for Na+-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelBSt have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelBSt, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na+-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelBSt is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na+-coupled MFS transporters.

Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers.

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.

SRPassing Co-translational Targeting: The Role of the Signal Recognition Particle in Protein Targeting and mRNA Protection.

Signal recognition particle (SRP) is an RNA and protein complex that exists in all domains of life. It consists of one protein and one noncoding RNA in some bacteria. It is more complex in eukaryotes and consists of six proteins and one noncoding RNA in mammals. In the eukaryotic cytoplasm, SRP co-translationally targets proteins to the endoplasmic reticulum and prevents misfolding and aggregation of the secretory proteins in the cytoplasm. It was demonstrated recently that SRP also possesses an earlier unknown function, the protection of mRNAs of secretory proteins from degradation. In this review, we analyze the progress in studies of SRPs from different organisms, SRP biogenesis, its structure, and function in protein targeting and mRNA protection.

Translational Control of Secretory Proteins in Health and Disease.

Secretory proteins are synthesized in a form of precursors with additional sequences at their N-terminal ends called signal peptides. The signal peptides are recognized co-translationally by signal recognition particle (SRP). This interaction leads to targeting to the endoplasmic reticulum (ER) membrane and translocation of the nascent chains into the ER lumen. It was demonstrated recently that in addition to a targeting function, SRP has a novel role in protection of secretory protein mRNAs from degradation. It was also found that the quality of secretory proteins is controlled by the recently discovered Regulation of Aberrant Protein Production (RAPP) pathway. RAPP monitors interactions of polypeptide nascent chains during their synthesis on the ribosomes and specifically degrades their mRNAs if these interactions are abolished due to mutations in the nascent chains or defects in the targeting factor. It was demonstrated that pathological RAPP activation is one of the molecular mechanisms of human diseases associated with defects in the secretory proteins. In this review, we discuss recent progress in understanding of translational control of secretory protein biogenesis on the ribosome and pathological consequences of its dysregulation in human diseases.

Structural and functional characterization of protein-lipid interactions of the Salmonella typhimurium melibiose transporter MelB.

BACKGROUND: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein. RESULTS: Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2-3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. CONCLUSIONS: (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.

Resorcinarene-Based Facial Glycosides: Implication of Detergent Flexibility on Membrane-Protein Stability.

As a membrane-mimetic system, detergent micelles are popularly used to extract membrane proteins from lipid environments and to maintain their solubility and stability in an aqueous medium. However, many membrane proteins encapsulated in conventional detergents tend to undergo structural degradation during extraction and purification, thus necessitating the development of new agents with enhanced properties. In the current study, two classes of new amphiphiles are introduced, resorcinarene-based glucoside and maltoside amphiphiles (designated RGAs and RMAs, respectively), for which the alkyl chains are facially segregated from the carbohydrate head groups. Of these facial amphiphiles, two RGAs (RGA-C11 and RGA-C13) conferred markedly enhanced stability to four tested membrane proteins compared to a gold-standard conventional detergent. The relatively high water solubility and micellar stability of the RGAs compared to the RMAs, along with their generally favourable behaviours for membrane protein stabilisation described here, are likely to be, at least in part, a result of the high conformational flexibility of these glucosides. This study suggests that flexibility could be an important factor in determining the suitability of new detergents for membrane protein studies.

Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies.

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy.

Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene-cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously developed xylene-linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein-coupled receptor (GPCR), some agents such as MGA-C13 and MGA-C14 resulted in markedly enhanced stability of membrane proteins compared to both DDM and the XMAs. This favourable behaviour is due likely to the increased hydrophobic density provided by the extra alkyl chain. Thus, this study not only describes new glucoside agents with potential for membrane protein research, but also introduces a new detergent design principle for future development.

On the role of TolC in multidrug efflux: the function and assembly of AcrAB-TolC tolerate significant depletion of intracellular TolC protein.

TolC channel provides a route for the expelled drugs and toxins to cross the outer membrane of Escherichia coli. The puzzling feature of TolC structure is that the periplasmic entrance of the channel is closed by dense packing of 12 α-helices. Efflux pumps exemplified by AcrAB are proposed to drive the opening of TolC channel. How interactions with AcrAB promote the close-to-open transition in TolC remains unclear. In this study, we investigated in vivo the functional and physical interactions of AcrAB with the closed TolC and its conformer opened by mutations in the periplasmic entrance. We found that the two conformers of TolC are readily distinguishable in vivo by characteristic drug susceptibility, thiol modification and proteolytic profiles. However, these profiles of TolC variants respond neither to the in vivo stoichiometry of AcrAB:TolC nor to the presence of vancomycin, which is used often to assess the permeability of TolC channel. We further found that the activity and assembly of AcrAB-TolC tolerates significant changes in amounts of TolC and that only a small fraction of intracellular TolC is likely used to support efflux needs of E. coli. Our findings explain why TolC is not a good target for inhibition of multidrug efflux.

Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability.

Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.

Molecular phenotypes segregate missense mutations in SLC13A5 Epilepsy.

The sodium-coupled citrate transporter (NaCT, SLC13A5) mediates citrate uptake across the plasma membrane via an inward Na + gradient. Mutations in SLC13A5 cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5 Epilepsy) due to impaired citrate uptake in neurons. Despite clinical identification of disease-causing mutations, underlying mechanisms and cures remain elusive. We mechanistically classify the molecular phenotypes of six mutations. C50R, T142M, and T227M exhibit impaired citrate transport despite normal expression at the cell surface. G219R, S427L, and L488P are hampered by low protein expression, ER retention, and reduced transport. Mutants' mRNA levels resemble wildtype, suggesting post-translational defects. Class II mutations display immature core-glycosylation and shortened half-lives, indicating protein folding defects. These experiments provide a comprehensive understanding of the mutation's defects in SLC13A5 Epilepsy at the biochemical and molecular level and shed light into the trafficking pathway(s) of NaCT. The two classes of mutations will require fundamentally different treatment approaches to either restore transport function, or enable correction of protein folding defects. Summary: Loss-of-function mutations in the SLC13A5 causes SLC13A5-Epilepsy, a devastating disease characterized by neonatal epilepsy. Currently no cure is available. We clarify the molecular-level defects to guide future developments for phenotype-specific treatment of disease-causing mutations.

Association of Blood NK Cell Phenotype with the Severity of Liver Fibrosis in Patients with Chronic Viral Hepatitis C with Genotype 1 or 3.

BACKGROUND: NK cells phenotype and functional state in different genotypes of chronic viral hepatitis C (CVHC), depending on liver fibrosis severity, have not been sufficiently studied, which limits the possibilities for the development of pathology therapy. METHODS: The CVHC diagnosis was based on the EASL recommendations (2018). Clinical examination with liver elastometry was performed in 297 patients with genotype 1 and in 231 patients with genotype 3 CVHC. The blood NK cells phenotype was determined by flow cytometry in 74 individuals with genotype 1 and in 69 individuals with genotype 3 CVHC. RESULTS: The frequency of METAVIR liver fibrosis stages F3-F4 was 32.5% in individuals with genotype 3, and 20.5% in individuals with genotype 1 CVHC (p = 0.003). In patients with both genotype 1 and genotype 3 CVHC, a decrease in the total number of blood NK cells, CD56brightCD16+ NK cells and an increase in the proportion of CD56dimCD16+ NK cells, CD94+ and CD38 + CD73+ NK cells were registered in patients with fibrosis stage F3-F4 by METAVIR in comparison with persons with METAVIR fibrosis stage F0-F1. CONCLUSIONS: In patients with both genotype 1 and genotype 3 CVHC, an imbalance in the ratio between cytokine-producing and cytotoxic NK cells and an increase in the content of NK cells that express inhibitory molecules were determined in patients with severe liver fibrosis.

Hydrocarbons in the water and bottom sediments of Sivash Bay (the Azov Sea) during its salinization.

Sivash Bay is a unique hypersaline lagoon located in the northern part of the Crimean Peninsula. In 2014, due to political events in connection with the closure of the North Crimean Canal, the inflow of fresh water to Sivash Bay has been significantly reduced. As a result, there has been a steady increase in salinity since 2014 to the present. The main purpose of this work was to determine the spatial distribution and qualitative composition of hydrocarbons (aliphatic hydrocarbons, n-alkanes, polycyclic aromatic hydrocarbons) in the water and bottom sediments of the hypersaline Sivash Bay under increasing water salinity. The analysis of the physico-chemical parameters of Sivash Bay in 2020 showed the continued salinization and change of physico-chemical conditions of the lagoon. At the same time, spatially, the change in salinity affected only the total content and qualitative composition of hydrocarbons in the water. The content of the studied classes of hydrocarbons in the bottom sediments did not demonstrate a reliable correlation with the concentration of salts. There was also no statistically significant dependence of Eh and pH of bottom sediments on salinity. In accordance with the composition of n-alkanes and polyaromatic hydrocarbons, as well as on the basis of PCA analysis, it is possible to make a conclusion on natural, mainly autochthonous, sources of this class of substances and low toxicity of bottom sediments of the bay. Low concentrations and composition of hydrocarbons indicate an insignificant input of pollutants of anthropogenic origin in Sivash Bay during its salinization.

Translational reprogramming as a driver of antimony-drug resistance in Leishmania.

Leishmania is a unicellular protozoan that has a limited transcriptional control and mostly uses post-transcriptional regulation of gene expression, although the molecular mechanisms of the process are still poorly understood. Treatments of leishmaniasis, pathologies associated with Leishmania infections, are limited due to drug resistance. Here, we report dramatic differences in mRNA translation in antimony drug-resistant and sensitive strains at the full translatome level. The major differences (2431 differentially translated transcripts) were demonstrated in the absence of the drug pressure supporting that complex preemptive adaptations are needed to efficiently compensate for the loss of biological fitness once they are exposed to the antimony. In contrast, drug-resistant parasites exposed to antimony activated a highly selective translation of only 156 transcripts. This selective mRNA translation is associated with surface protein rearrangement, optimized energy metabolism, amastins upregulation, and improved antioxidant response. We propose a novel model that establishes translational control as a major driver of antimony-resistant phenotypes in Leishmania.

Pathogenic signal peptide variants in the human genome.

Secreted and membrane proteins represent a third of all cellular proteins and contain N-terminal signal peptides that are required for protein targeting to endoplasmic reticulum (ER). Mutations in signal peptides affect protein targeting, translocation, processing, and stability, and are associated with human diseases. However, only a few of them have been identified or characterized. In this report, we identified pathogenic signal peptide variants across the human genome using bioinformatic analyses and predicted the molecular mechanisms of their pathology. We recovered more than 65 thousand signal peptide mutations, over 11 thousand we classified as pathogenic, and proposed framework for distinction of their molecular mechanisms. The pathogenic mutations affect over 3.3 thousand genes coding for secreted and membrane proteins. Most pathogenic mutations alter the signal peptide hydrophobic core, a critical recognition region for the signal recognition particle, potentially activating the Regulation of Aberrant Protein Production (RAPP) quality control and specific mRNA degradation. The remaining pathogenic variants (about 25%) alter either the N-terminal region or signal peptidase processing site that can result in translocation deficiencies at the ER membrane or inhibit protein processing. This work provides a conceptual framework for the identification of mutations across the genome and their connection with human disease.

The Enrichment of miRNA-Targeted mRNAs in Translationally Less Active over More Active Polysomes.

miRNAs moderately inhibit the translation and enhance the degradation of their target mRNAs via cognate binding sites located predominantly in the 3'-untranslated regions (UTR). Paradoxically, miRNA targets are also polysome-associated. We studied the polysome association by the comparative translationally less-active light- and more-active heavy-polysome profiling of a wild type (WT) human cell line and its isogenic mutant (MT) with a disrupted DICER1 gene and, thus, mature miRNA production. As expected, the open reading frame (ORF) length is a major determinant of light- to heavy-polysome mRNA abundance ratios, but is rendered less powerful in WT than in MT cells by miRNA-regulatory activities. We also observed that miRNAs tend to target mRNAs with longer ORFs, and that adjusting the mRNA abundance ratio with the ORF length improves its correlation with the 3'-UTR miRNA-binding-site count. In WT cells, miRNA-targeted mRNAs exhibit higher abundance in light relative to heavy polysomes, i.e., light-polysome enrichment. In MT cells, the DICER1 disruption not only significantly abrogated the light-polysome enrichment, but also narrowed the mRNA abundance ratio value range. Additionally, the abrogation of the enrichment due to the DICER1 gene disruption, i.e., the decreases of the ORF-length-adjusted mRNA abundance ratio from WT to MT cells, exhibits a nearly perfect linear correlation with the 3'-UTR binding-site count. Transcription factors and protein kinases are the top two most enriched mRNA groups. Taken together, the results provide evidence for the light-polysome enrichment of miRNA-targeted mRNAs to reconcile polysome association and moderate translation inhibition, and that ORF length is an important, though currently under-appreciated, transcriptome regulation parameter.

Effect of an NGR Peptide on the Efficacy of the Doxorubicin Phospholipid Delivery System.

This study is a continuation of an investigation into the effect of a targeted component, a peptide with an NGR, on the properties of the previously developed doxorubicin phospholipid delivery system. The NGR peptide has an affinity for aminopeptidase N (known as the CD13 marker on the membrane surface of tumor cells) and has been extensively used to target drug delivery systems. This article presents the results of a study investigating the physical properties of the phospholipid composition with and without the peptide chain: particle size, zeta potential, stability in fluids, and dependence of doxorubicin release from nanoparticles at different pH levels (5.0, 6.5, 7.4). The cytotoxic effect of the compositions has also been shown to depend on the dose of the drug used for incubation, the presence of the targeted component in the composition, and the time of incubation time of the substances. There was a significant difference in the cytotoxic effect on HT-1080 (CD13-positive) and MCF-7 (CD13-negative) cells. Cell death pathway analysis has shown that death occurred mainly by apoptosis. We also present data on the effect of doxorubicin embedded in phospholipid nanoparticles with the targeted peptide on DNA assessed by differential pulse voltammetry, the mechanism of action being electrostatic interactions. The interactions of native dsDNA with doxorubicin encapsulated in phospholipid nanoparticles with the targeted peptide were studied electrochemically by differential pulse voltammetry. Here, we have highlighted that the targeted peptide in the doxorubicin composition moved specific interaction of the drug with dsDNA from intercalative mode to electrostatic interactions.