Long-Term Cryopreservation May Cause Genomic Instability and the Premature Senescence of Cells.

Cryopreservation is an essential step for utilizing various cell types for biological research and medical purposes. At the same time, there is a lack of data on the effect of cryopreservation, especially when prolonged, on the karyotype of cells. In the present work, we analyzed the genetic stability of cells subjected to a cryopreservation procedure. The objects were immortalized Chinese hamster lung fibroblasts (CHL V-79 RJK line) and human endometrial mesenchymal stem/stromal cells (eMSCs). We showed that short-term cryopreservation in liquid nitrogen for up to 6 months did not affect the karyotype stability of CHL V-79 RJK and eMSCs. On the contrary, karyotyping of G-banded metaphase chromosomes in cells underwent 10-year cryopreservation, which revealed genomic instability in both cell lines associated with the variability of chromosome number in cells, random chromosomal rearrangements, and condensation disorder in homologs. In addition, we found out that long-term cryopreservation of eMSCs does not affect the expression of their typical surface markers and morphology, but results in a significant reduction in proliferative potential and early manifestation of cellular senescence features upon eMSCs culturing. Thus, we concluded that the long-term cryopreservation of cells of different types and biological origin can lead to irreversible changes of their karyotype and acceleration of cellular senescence.

HyPer as a tool to determine the reductive activity in cellular compartments.

A multitude of cellular metabolic and regulatory processes rely on controlled thiol reduction and oxidation mechanisms. Due to our aerobic environment, research preferentially focuses on oxidation processes, leading to limited tools tailored for investigating cellular reduction. Here, we advocate for repurposing HyPer1, initially designed as a fluorescent probe for H2O2 levels, as a tool to measure the reductive power in various cellular compartments. The response of HyPer1 depends on kinetics between thiol oxidation and reduction in its OxyR sensing domain. Here, we focused on the reduction half-reaction of HyPer1. We showed that HyPer1 primarily relies on Trx/TrxR-mediated reduction in the cytosol and nucleus, characterized by a second order rate constant of 5.8 × 102 M-1s-1. On the other hand, within the mitochondria, HyPer1 is predominantly reduced by glutathione (GSH). The GSH-mediated reduction rate constant is 1.8 M-1s-1. Using human leukemia K-562 cells after a brief oxidative exposure, we quantified the compartmentalized Trx/TrxR and GSH-dependent reductive activity using HyPer1. Notably, the recovery period for mitochondrial HyPer1 was twice as long compared to cytosolic and nuclear HyPer1. After exploring various human cells, we revealed a potent cytosolic Trx/TrxR pathway, particularly pronounced in cancer cell lines such as K-562 and HeLa. In conclusion, our study demonstrates that HyPer1 can be harnessed as a robust tool for assessing compartmentalized reduction activity in cells following oxidative stress.

Expected and Unexpected Effects of Pharmacological Antioxidants.

In this review, we have collected the existing data on the bioactivity of antioxidants (N-acetylcysteine, polyphenols, vitamin C) which are traditionally used in experimental biology and, in some cases, in the clinic. Presented data show that, despite the capacity of these substances to scavenge peroxides and free radicals in cell-free systems, their ability to exhibit these properties in vivo, upon pharmacological supplementation, has not been confirmed so far. Their cytoprotective activity is explained mainly by the ability not to suppress, but to activate multiple redox pathways, which causes biphasic hormetic responses and highly pleiotropic effects in cells. N-acetylcysteine, polyphenols, and vitamin C affect redox homeostasis by generating low-molecular-weight redox-active compounds (H2O2 or H2S), known for their ability to stimulate cellular endogenous antioxidant defense and promote cytoprotection at low concentrations but exert deleterious effects at high concentrations. Moreover, the activity of antioxidants strongly depends on the biological context and mode of their application. We show here that considering the biphasic and context-dependent response of cells on the pleiotropic action of antioxidants can help explain many of the conflicting results obtained in basic and applied research and build a more logical strategy for their use.

Inhibition of thioredoxin reductase activity reduces the antioxidant defense capacity of human pluripotent stem cells under conditions of mild but not severe oxidative stress.

Pro-oxidative shift in redox balance, usually termed as "oxidative stress", can lead to different cell responses depending on its intensity. Excessive accumulation of reactive oxygen species ("oxidative distress") can cause DNA, lipid and protein damage. Physiological oxidative stimulus ("oxidative eustress"), in turn, can favor cell proliferation and differentiation - the processes of paramount importance primarily for stem cells. Functions of antioxidant enzymes in cells is currently a focus of intense research, however the role of different antioxidant pathways in pluripotent cell responses to oxidative distress/eustress is still under investigation. In this study, we assessed the contribution of the thioredoxin reductase (TrxR)-dependent pathways to maintaining the redox homeostasis in human induced pluripotent stem cells and their differentiated progeny cells under basal conditions and under conditions of oxidative stress of varying intensity. Employing the genetically encoded H2O2 biosensor cyto-HyPer and two inhibitors of thioredoxin reductase (auranofin and Tri-1), we show that the reduced activity of TrxR-dependent enzymatic systems leads to the non-cytotoxic disruption of thiol-disulfide metabolism in the cytoplasm of both pluripotent and differentiated cells under basal conditions. Quantifying the cytoplasmic concentrations of peroxide establishing in H2O2-stressed cells, we demonstrate that TrxR-dependent pathways contribute to the antioxidant activity in the cell cytoplasm under conditions of mild but not severe oxidative stress in both cell lines tested. The observed effects may testify about a conservative role of the TrxR-controlled enzymatic systems manifested as a response to physiological redox stimuli rather than a protection against the severe oxidative stress.

Paracrine and Autocrine Effects of VEGF Are Enhanced in Human eMSC Spheroids.

The mechanisms underlying the therapeutic potential of MSCs are the focus of intense research. We studied human MSCs isolated from desquamated endometrium (eMSCs), which, as previously shown, have high regenerative potential in various disease models. The aim was to evaluate the role of secreted VEGF in stimulating angiogenesis and maintaining eMSC viability and migration, which is important for improving the therapeutic properties of MSCs. We compared three eMSC cultures differing in the level of VEGF secretion: 3D spheroids, monolayer eMSCs, and monolayer eMSCs with VEGF knockdown. Spheroid eMSCs produced higher amounts of VEGF and had the strongest paracrine effect on HUVEC. eMSCs with VEGF knockdown did not stimulate angiogenesis. Monolayered eMSCs expressed VEGFR1, while spheroid eMSCs expressed both VEGFR1 and VEGFR2 receptors. The knockdown of VEGF caused a significant decrease in the viability and migration of eMSCs. eMSCs from 3D spheroids enhanced proliferation and migration in response to exogenous VEGF, in contrast to monolayered eMSCs. Our results suggest that the VEGF-VEGFR1 loop appears to be autocrine-involved in maintaining the viability of eMSCs, and VEGFR2 expression enhances their response to exogenous VEGF, so the angiogenic potential of eMSC can be up- or downregulated by intrinsic VEGF signals.

Resistance to H2O2-induced oxidative stress in human cells of different phenotypes.

Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (H2O2) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic H2O2 concentrations under conditions of H2O2-induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells - all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes - mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous H2O2, we showed that at low oxidative loads (<50 µM of H2O2) the gradient depended on extracellular H2O2 concentration. At high loads (>50 µM of H2O2), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H2O2 gradient on the oxidative load in human cells. At high H2O2 concentrations, the cytoplasmic H2O2 level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the H2O2-detoxification processes is inherent to a period of early human development.

Induction of Premature Cell Senescence Stimulated by High Doses of Antioxidants Is Mediated by Endoplasmic Reticulum Stress.

In our previous study, we found that high doses of several substances with antioxidant capacities (Tempol, resveratrol, diphenyleneiodonium) can cause genotoxic stress and induce premature senescence in the human mesenchymal stem cells (MSCs). Here, using whole-transcriptome analysis, we revealed the signs of endoplasmic reticulum stress and unfolded protein response (UPR) in MSCs stressed with Tempol and resveratrol. In addition, we found the upregulation of genes, coding the UPR downstream target APC/C, and E3 ubiquitin ligase that regulate the stability of cell cycle proteins. We performed the molecular analysis, which further confirmed the untimely degradation of APC/C targets (cyclin A, geminin, and Emi1) in MSCs treated with antioxidants. Human fibroblasts responded to antioxidant applications similarly. We conclude that endoplasmic reticulum stress and impaired DNA synthesis regulation can be considered as potential triggers of cell damage and premature senescence stimulated by high-dose antioxidant treatments.

Redox Homeostasis and Regulation in Pluripotent Stem Cells: Uniqueness or Versatility?

Pluripotent stem cells (PSCs) hold great potential both in studies on developmental biology and clinical practice. Mitochondrial metabolism that encompasses pathways that generate ATP and produce ROS significantly differs between PSCs and somatic cells. Correspondingly, for quite a long time it was believed that the redox homeostasis in PSCs is also highly specific due to the hypoxic niche of their origin-within the pre-implantation blastocyst. However, recent research showed that redox parameters of cultivated PSCs have much in common with that of their differentiated progeny cells. Moreover, it has been proven that, similar to somatic cells, maintaining the physiological ROS level is critical for the regulation of PSC identity, proliferation, differentiation, and de-differentiation. In this review, we aimed to summarize the studies of redox metabolism and signaling in PSCs to compare the redox profiles of pluripotent and differentiated somatic cells. We collected evidence that PSCs possess metabolic plasticity and are able to adapt to both hypoxia and normoxia, that pluripotency is not strictly associated with anaerobic conditions, and that cellular redox homeostasis is similar in PSCs and many other somatic cells under in vitro conditions that may be explained by the high conservatism of the redox regulation system.

Cell cycle-coupled changes in the level of reactive oxygen species support the proliferation of human pluripotent stem cells.

The study of proliferation regulation in human pluripotent stem cells is crucial to gain insights into understanding the physiology of these cells. However, redox regulation of the pluripotent cell cycle remains largely unexplored. Here, using human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs), we demonstrate that the level of reactive oxygen species (ROS) in pluripotent cells oscillates in accordance with the cell cycle progression with the peak occurring at transition from S to G2 /M phase of the cycle. A decrease of this level by antioxidants leads to hindered S-phase initiation and progression but does not affect the early-G1 -phase or mitosis. Cells exposed to antioxidants in the early-G1 -phase accumulate the phosphorylated retinoblastoma protein and overcome the restriction point but are unable to accumulate the main regulators of the S phase-CYCLIN A and GEMININ. Based on the previous findings that CYCLIN A stability is affected by redox homeostasis disturbances in somatic cells, we compared the responses to antioxidant treatments in hESCs and in their differentiated fibroblast-like progeny cells (difESCs). In difESCs, similar to hESCs, a decrease in ROS level results in the disruption of S-phase initiation accompanied by a deficiency of the CYCLIN A level. Moreover, in antioxidant-treated cells, we revealed the accumulation of DNA breaks, which was accompanied by activation of the apoptosis program in pluripotent cells. Thus, we conclude that maintaining the physiological ROS level is essential for promotion of proliferation and accurate DNA synthesis in pluripotent cells and their differentiated descendants.

Outcomes of Deferoxamine Action on H2O2-Induced Growth Inhibition and Senescence Progression of Human Endometrial Stem Cells.

Mesenchymal stem cells (MSCs) are broadly applied in regenerative therapy to replace cells that are lost or impaired during disease. The low survival rate of MSCs after transplantation is one of the major limitations heavily influencing the success of the therapy. Unfavorable microenvironments with inflammation and oxidative stress in the damaged regions contribute to MSCs loss. Most of the strategies developed to overcome this obstacle are aimed to prevent stress-induced apoptosis, with little attention paid to senescence-another common stress reaction of MSCs. Here, we proposed the strategy to prevent oxidative stress-induced senescence of human endometrial stem cells (hMESCs) based on deferoxamine (DFO) application. DFO prevented DNA damage and stress-induced senescence of hMESCs, as evidenced by reduced levels of reactive oxygen species, lipofuscin, cyclin D1, decreased SA-ß-Gal activity, and improved mitochondrial function. Additionally, DFO caused accumulation of HIF-1α, which may contribute to the survival of H2O2-treated cells. Importantly, cells that escaped senescence due to DFO preconditioning preserved all the properties of the initial hMESCs. Therefore, once protecting cells from oxidative damage, DFO did not alter further hMESCs functioning. The data obtained may become the important prerequisite for development of a new strategy in regenerative therapy based on MSCs preconditioning using DFO.

Generation of Therapeutically Potent Spheroids from Human Endometrial Mesenchymal Stem/Stromal Cells.

Endometrial mesenchymal stem/stromal cells (eMSCs) hold great promise in bioengineering and regenerative medicine due to their high expansion potential, unique immunosuppressive properties and multilineage differentiation capacity. Usually, eMSCs are maintained and applied as a monolayer culture. Recently, using animal models with endometrial and skin defects, we showed that formation of multicellular aggregates known as spheroids from eMSCs enhances their tissue repair capabilities. In this work, we refined a method of spheroid formation, which makes it possible to obtain well-formed aggregates with a narrow size distribution both at early eMSC passages and after prolonged cultivation. The use of serum-free media allows this method to be used for the production of spheroids for clinical purposes. Wound healing experiments on animals confirmed the high therapeutic potency of the produced eMSC spheroids in comparison to the monolayer eMSC culture.

Impact of Polyallylamine Hydrochloride on Gene Expression and Karyotypic Stability of Multidrug Resistant Transformed Cells.

The synthetic polymer, polyallylamine hydrochloride (PAA), is found in a variety of applications in biotechnology and medicine. It is used in gene and siRNA transfer, to form microcapsules for targeted drug delivery to damaged and tumor cells. Conventional chemotherapy often does not kill all cancer cells and leads to multidrug resistance (MDR). Until recently, studies of the effects of PAA on cells have mainly focused on their morphological and genetic characteristics immediately or several hours after exposure to the polymer. The properties of the cell progeny which survived the sublethal effects of PAA and resumed their proliferation, were not monitored. The present study demonstrated that treatment of immortalized Chinese hamster cells CHLV-79 RJK sensitive (RJK) and resistant (RJKEB) to ethidium bromide (EB) with cytotoxic doses of PAA, selected cells with increased karyotypic instability, were accompanied by changes in the expression of p53 genes c-fos, topo2-α, hsp90, hsc70. These changes did not contribute to the progression of MDR, accompanied by the increased sensitivity of these cells to the toxic effects of doxorubicin (DOX). Our results showed that PAA does not increase the oncogenic potential of immortalized cells and confirmed that it can be used for intracellular drug delivery for anticancer therapy.

Three-Dimensional Compaction Switches Stress Response Programs and Enhances Therapeutic Efficacy of Endometrial Mesenchymal Stem/Stromal Cells.

Mesenchymal stem cells are currently tested as a promising tool for the treatment of a wide range of human diseases. Enhanced therapeutic potential of spheroids formed from these cells has been proved in numerous studies, however, the fundamental basics of this effect are still being discussed. In this work, we showed that endometrial mesenchymal stem/stromal cells (eMSCs) assembled in spheroids possess a higher therapeutic efficacy compared to cells grown in monolayer in the treatment of the defects that are non-specific for eMSC tissue origin - skin wounds. With the purpose to elucidate the possible causes of superior spheroid potency, we compared the tolerance of eMSC cultivated in spheres and monolayer to the stress insults. Using genetically encoded hydrogen peroxide biosensor HyPer, we showed that three-dimensional configuration (3D) helped to shield the inner cell layers of spheroid from the external H2O2-induced oxidative stress. However, the viability of oxidatively damaged eMSCs in spheroids appeared to be much lower than that of monolayer cells. An extensive analysis, which included administration of heat shock and irradiation stress, revealed that cells in spheroids damaged by stress factors activate the apoptosis program, while in monolayer cells stress-induced premature senescence is developed. We found that basal down-regulation of anti-apoptotic and autophagy-related genes provides the possible molecular basis of the high commitment of eMSCs cultured in 3D to apoptosis. We conclude that predisposition to apoptosis provides the programmed elimination of damaged cells and contributes to the transplant safety of spheroids. In addition, to investigate the role of paracrine secretion in the wound healing potency of spheroids, we exploited the in vitro wound model (scratch assay) and found that culture medium conditioned by eMSC spheroids accelerates the migration of adherent cells. We showed that 3D eMSCs upregulate transcriptional activator, hypoxia-inducible factor (HIF)-1, and secret ten-fold more HIF-1-inducible pro-angiogenic factor VEGF (vascular endothelial growth factor) than monolayer cells. Taken together, these findings indicate that enhanced secretory activity can promote wound healing potential of eMSC spheroids and that cultivation in the 3D cell environment alters eMSC vital programs and therapeutic efficacy.

Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function.

Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H2O2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts.

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer.

In this study, we propose a method for quantification of average hydrogen peroxide concentration within a living cell that is based on the use of genetically encoded H2O2 biosensor HyPer. The method utilizes flow cytometric measurements of HyPer fluorescence in H2O2-exposed cells to analyze the biosensor oxidation kinetics. Fitting the experimental curves with kinetic equations allows determining the rate constants of HyPer oxidation/reduction which are used further for the calculation of peroxide concentrations in the cells of interest both in the presence and absence of external H2O2. Applying this method to K562 cells, we have estimated the gradient as about 390-fold between the extracellular and intracellular level of exogenous H2O2 in cells exposed to the micromole doses of peroxide, as well as the average basal level of H2O2 in the cytosol of undisturbed cells ( [Formula: see text] ). The method can be extended to other H2O2-sensitive redox probes or to procedures in which, rather than adding external peroxide, intracellular production of peroxide is triggered, providing a tool to quantitate not only basal average H2O2 concentrations but also the concentration of peroxide build up in the vicinity of redox probes.

Cell Cycle-Dependent Expression of Bk Channels in Human Mesenchymal Endometrial Stem Cells.

The study of ion channels in stem cells provides important information about their role in stem cell fate. Previously we have identified the activity of calcium-activated potassium channels of big conductance (BK channels) in human endometrium-derived mesenchymal stem cells (eMSCs). BK channels could have significant impact into signaling processes by modulating membrane potential. The membrane potential and ionic permeability dynamically changes during cycle transitions. Here, we aimed at verification of the role of BK channels as potassium transporting pathway regulating cell cycle passageway of eMSCs. The functional expression of native BK channels was confirmed by patch-clamp and immunocytochemistry. In non-synchronized cells immunofluorescent analysis revealed BK-positive and BK-negative stained eMSCs. Using cell synchronization, we found that the presence of BK channels in plasma membrane was cell cycle-dependent and significantly decreased in G2M phase. However, the study of cell cycle progression in presence of selective BK channel inhibitors showed no effect of pore blockers on cycle transitions. Thus, BK channel-mediated K+ transport is not critical for the fundamental mechanism of passageway through cell cycle of eMSCs. At the same time, the dynamics of the presence of BK channels on plasma membrane of eMSCs can be a novel indicator of cellular proliferation.

Human mesenchymal stem cells in spheroids improve fertility in model animals with damaged endometrium.

BACKGROUND: Asherman's syndrome (AS) is one of the gynecological disorders caused by the destruction of the endometrium. For some cases of AS available surgical methods and hormonal therapy are ineffective. Stem cell transplantation may offer a potential alternative for AS cure. METHODS: Human endometrial mesenchymal stem cells (eMSC) organized in spheroids were transplanted in rats with damaged endometrium modeled on AS. Treatment response was defined as pregnancy outcome and litter size. RESULTS: Application of eMSC in spheroids significantly improved the rat fertility with the AS model. eMSC organized in spheroids retain all properties of eMSC in monolayer: growth characteristics, expression of CD markers, and differentiation potential. Synthesis of angiogenic and anti-inflammatory factors drastically increased in eMSC assembled into spheroids. CONCLUSIONS: Human endometrial mesenchymal stem cells (eMSC) can be successfully applied for Asherman's syndrome (AS) treatment in the rat model. eMSC organized in spheroids were more therapeutically effective than the cells in monolayer. After transplantation of eMSC in spheroids the pregnancy outcome and litter size in rats with AS was higher than in rats that received autologous rat bone marrow cells. It suggests the therapeutic plausibility of heterologous eMSC in case of failure to use autologous cells.

Therapeutic doses of doxorubicin induce premature senescence of human mesenchymal stem cells derived from menstrual blood, bone marrow and adipose tissue.

Doxorubicin (Dox) is an effective anticancer drug with known activity against a wide spectrum of malignancies, hematologic malignancies in particular. Despite extensive clinical use, the mechanisms of its side effects and negative action on normal cells remain under study. The aim of this study was to investigate the effect of Dox on cultured human mesenchymal stem cells (MSCs) derived from menstrual blood (eMSCs), bone marrow (BMSCs) and adipose tissue (AMSCs). Dox treatment in high doses decreased the survival of MSCs in a dose-dependent manner. Clinically relevant low doses of Dox induced premature senescence of eMSCs, BMSCs and AMSCs, but did not kill the cells. Dox caused cell cycle arrest and formation of γ-H2AX foci, and increased the number of SA-ß-gal-positive cells. BMSCs entered premature senescence earlier than other MSCs. It has been reported that neural-like cells differentiated from MSCs of various origins are more sensitive to Dox than their parent cells. Dox-treated differentiated MSCs exhibited lower viability and earlier generation of γ-H2AX foci. Dox administration inhibited secretory activity in neural-like cells. These findings suggest that a clinically relevant Dox dose damages cultured MSCs, inducing their premature senescence. MSCs are more resistant to this damage than differentiated cells.

Proteomic analysis of affinity-purified extracellular proteasomes reveals exclusively 20S complexes.

Proteasome-mediated proteolysis is important for many basic cellular processes. In addition to their functions in the cell, proteasomes have been found in physiological fluids of both healthy and diseased humans including cancer patients. Higher levels of these proteasomes are associated with higher cancer burden and stage. The etiology and functions of these proteasomes, referred to as circulating, plasmatic, or extracellular proteasomes (ex-PSs), are unclear. Here we show that human cancer cell lines, as well as human endometrium-derived mesenchymal stem cells (hMESCs), release proteasome complexes into culture medium (CM). To define ex-PS composition, we have affinity purified them from CM conditioned by human leukemia cell line K562. Using matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), we have identified core 20S proteasome subunits and a set of 15 proteasome-interacting proteins (PIPs), all previously described as exosome cargo proteins. Three of them, PPIase A, aldolase A, and transferrin, have never been reported as PIPs. The study provides compelling arguments that ex-PSs do not contain 19S or PA200 regulatory particles and are represented exclusively by the 20S complex.

Origin of anti-tumor activity of the cysteine-containing GO peptides and further optimization of their cytotoxic properties.

Antitumor GO peptides have been designed as dimerization inhibitors of prominent oncoprotein mucin 1. In this study we demonstrate that activity of GO peptides is independent of the level of cellular expression of mucin 1. Furthermore, these peptides prove to be broadly cytotoxic, causing cell death also in normal cells such as dermal fibroblasts and endometrial mesenchymal stem cells. To explore molecular mechanism of their cytotoxicity, we have designed and tested a number of new peptide sequences containing the key CxC or CxxC motifs. Of note, these sequences bear no similarity to mucin 1 except that they also contain a pair of proximal cysteines. Several of the new peptides turned out to be significantly more potent than their GO prototypes. The results suggest that cytotoxicity of these peptides stems from their (moderate) activity as disulfide oxidoreductases. It is expected that such peptides, which we have termed DO peptides, are involved in disulfide-dithiol exchange reaction, resulting in formation of adventitious disulfide bridges in cell proteins. In turn, this leads to a partial loss of protein function and rapid onset of apoptosis. We anticipate that coupling DO sequences with tumor-homing transduction domains can create a potentially valuable new class of tumoricidal peptides.