Annexins are a conserved family of calcium- and phospholipid-binding proteins. Despite their ubiquity and ancient origin, their physiological functions are not fully understood. Numerous studies have shown that the ectopic expression of some annexins can ameliorate the adverse effects of numerous abiotic stresses through modulation of reactive oxygen species accumulation and/or abscisic acid signaling. To gain further insight into the molecular mechanisms of this activity, we studied transgenic Arabidopsis thaliana (Arabidopsis) plants with increased expression of ANNEXIN1 (ATANN1) in the wild-type Col-0 lines (Col-OX) and in GO mutants (GO-OX) synchronously accumulating high levels of hydrogen peroxide in chloroplasts. Comprehensive biochemical and transcriptomic analyses led us to propose that ATANN1 modulates the turnover of ascorbate and glutathione, thereby affecting various redox-dependent processes, including photosynthesis, and phytohormonal signaling. Compared to the non-transgenic line, the overexpression of ATANN1 resulted in a two-to threefold increase in jasmonic acid accumulation in response to high light and oxidative stress in both genetic backgrounds analyzed (about 200 % compared to the control conditions in WT, while about 300-400 % in transgenic lines). This also affected salicylic acid-activated transcriptional reprogramming, although a slight up-regulation of this phytohormone occurred only in GO-OX. Overall, ATANN1 affects the integration of the SA and JA signaling pathways in such conditions. Our results provide compelling evidence that during stress, ATANN1 is involved in the regulation of redox homeostasis and the orchestration of diverse hormone-mediated signaling pathways, thereby fine-tuning the plant response to stress.

Epidemiological studies show that cardiovascular events related to platelet hyperactivity remain the leading causes of death among multiple sclerosis (MS) patients. Quantitative or structural changes of platelet cytoskeleton alter their morphology and function. Here, we demonstrated, for the first time, the structural changes in MS platelets that may be related to their hyperactivity. MS platelets were found to form large aggregates compared to control platelets. In contrast to the control, the images of overactivated, irregularly shaped MS platelets show changes in the cytoskeleton architecture, fragmented microtubule rings. Furthermore, MS platelets have long and numerous pseudopodia rich in actin filaments. We showed that MS platelets and megakaryocytes, overexpress β1-tubulin and β-actin mRNAs and proteins and have altered post-translational modification patterns. Moreover, we identified two previously undisclosed mutations in the gene encoding β1-tubulin in MS. We propose that the demonstrated structural changes of platelet cytoskeleton enhance their ability to adhere, aggregate, and degranulate fueling the risk of adverse cardiovascular events in MS.

In natural environments, bacteria often enter a state of anhydrobiosis due to water loss. Multiple studies have demonstrated that desiccation may lead to protein aggregation and glycation both in vivo and in vitro. However, the exact effects of water-loss-induced proteotoxic stress and the interplay between protein glycation and aggregation in bacteria remain elusive. Our studies revealed that protein aggregates formation in Escherichia coli started during desiccation and continued during the rehydration stage. The aggregates were enriched in proteins prone to liquid-liquid phase separation. Although it is known that glycation may induce protein aggregation in vitro, the aggregates formed in E. coli contained low levels of glycation products compared to the soluble protein fraction. Carnosine, glycine betaine and trehalose diminished the formation of protein aggregates and glycation products, resulting in increased E. coli viability. Notably, although high concentrations of glycine-betaine and trehalose significantly enhanced protein aggregation, glycation was still inhibited and E. coli cells survived desiccation better than bacteria grown without osmolytes. Taken together, our results suggest that the aggregates might play protective functions during early desiccation-rehydration stress. Moreover, it seems glycation rather than protein aggregation is the main cause of E. coli death upon desiccation-rehydration stress.

(1) Objective: The aim of this dynamic LC-MS (liquid chromatography and mass spectrometry) human platelet proteomic study was to identify the potential proteins candidates for biomarkers of acute ischemic stroke (AIS), their changes during the acute phase of stroke and to define potential novel drug targets. (2) Methods: A total of 32 patients (18–80 years old) were investigated that presented symptoms of AIS lasting less than 24 h from the onset, confirmed by neurological examination and/or new cerebral ischemia visualized in the CT (computed-tomography) scans. The analysis of platelet proteome was performed using LC-MS at baseline, and then on the third and seventh day from the onset of symptoms. The control group was demographically matched without any clinical signs of acute brain injury. (3) Results: The differences between platelets, at 24 h after first symptoms of stroke subjects and the control group included: β-amyloid A4 and amyloid-like protein 2, coactosin-like protein, thymidine phosphorylase 4 (TYMP-4), interferon regulatory factor 7 (IRF7), vitamin K-dependent protein S, histone proteins (H2A type 1 and 1-A, H2A types 2B and J, H2Av, -z, and -x), and platelet basic protein. The dynamic changes in the platelet protein concentration involved thrombospondin-1, thrombospondin-2, filamin A, B, and C. (4) Conclusions: This is the first human dynamic LC-MS proteomic study that differentiates platelet proteome in the acute phase of ischemic stroke in time series and compares the results with healthy controls. Identified proteins may be considered as future markers of ischemic stroke or therapeutic drug targets. Thymidine phosphorylase 4 (TYMP-4) holds promise as an interesting drug target in the management or prevention of ischemic stroke.

(1) Background: The aim of this dynamic-LC/MS-human-serum-proteomic-study was to identify potential proteins-candidates for biomarkers of acute ischemic stroke, their changes during acute phase of stroke and to define potential novel drug-targets. (2) Methods: A total of 32 patients (29–80 years) with acute ischemic stroke were enrolled to the study. The control group constituted 29 demographically-matched volunteers. Subjects with stroke presented clinical symptoms lasting no longer than 24 h, confirmed by neurological-examination and/or new cerebral ischemia visualized in the CT scans (computed tomography). The analysis of plasma proteome was performed using LC-MS (liquid chromatography–mass spectrometry). (3) Results: Ten proteins with significantly different serum concentrations between groups volunteers were: complement-factor-B, apolipoprotein-A-I, fibronectin, alpha-2-HS-glycoprotein, alpha-1B-glycoprotein, heat-shock-cognate-71kDa protein/heat-shock-related-70kDa-protein-2, thymidine phosphorylase-2, cytoplasmic-tryptophan-tRNA-ligase, ficolin-2, beta-Ala-His-dipeptidase. (4) Conclusions: This is the first dynamic LC-MS study performed on a clinical model which differentiates serum proteome of patients in acute phase of ischemic stroke in time series and compares to control group. Listed proteins should be considered as risk factors, markers of ischemic stroke or potential therapeutic targets. Further clinical validation might define their exact role in differential diagnostics, monitoring the course of the ischemic stroke or specifying them as novel drug targets.

Background: Dyslipidaemia is a major risk factor for atherosclerosis and cardiovascular diseases. The molecular mechanisms that translate dyslipidaemia into atherogenesis and reliable markers of its progression are yet to be fully elucidated. To address this issue, we conducted a comprehensive metabolomic and proteomic analysis in an experimental model of dyslipidaemia and in patients with familial hypercholesterolemia (FH). Methods: Liquid chromatography/mass spectrometry (LC/MS) and immunoassays were used to find out blood alterations at metabolite and protein levels in dyslipidaemic ApoE(-/-)/LDLR-/- mice and in FH patients to evaluate their human relevance. Results: We identified 15 metabolites (inhibitors and substrates of nitric oxide synthase (NOS), low-molecular-weight antioxidants (glutamine, taurine), homocysteine, methionine, 1-methylnicotinamide, alanine and hydroxyproline) and 9 proteins (C-reactive protein, proprotein convertase subtilisin/kexin type 9, apolipoprotein C-III, soluble intercellular adhesion molecule-1, angiotensinogen, paraoxonase-1, fetuin-B, vitamin K-dependent protein S and biglycan) that differentiated FH patients from healthy controls. Most of these changes were consistently found in dyslipidaemic mice and were further amplified if mice were fed an atherogenic (Western or low-carbohydrate, high-protein) diet. Conclusions: The alterations highlighted the involvement of an immune-inflammatory response system, oxidative stress, hyper-coagulation and impairment in the vascular function/regenerative capacity in response to dyslipidaemia that may also be directly engaged in development of atherosclerosis. Our study further identified potential biomarkers for an increased risk of atherosclerosis that may aid in clinical diagnosis or in the personalized treatment.

Objective: Dyslipidaemia is a major risk factor for myocardial infarction that is known to correlate with atherosclerosis in the coronary arteries. We sought to clarify whether metabolic alterations induced by dyslipidaemia in cardiomyocytes collectively constitute an alternative pathway that escalates myocardial injury. Methods: Dyslipidaemic apolipoprotein E and low-density lipoprotein receptor (ApoE/LDLR) double knockout (ApoE(-/-)/LDLR-/-) and wild- type C57BL/6 (WT) mice aged six months old were studied. Cardiac injury under reduced oxygen supply was evaluated by 5 min exposure to 5% oxygen in the breathing air under electrocardiogram (ECG) recording and with the assessment of troponin I release. To address the mechanisms LC/MS was used to analyse the cardiac proteome pattern or in vivo metabolism of stable isotope-labelled substrates and HPLC was applied to measure concentrations of cardiac high-energy phosphates. Furthermore, the effect of blocking fatty acid use with ranolazine on the substrate preference and cardiac hypoxic damage was studied in ApoE(-/-)/LDLR-/- mice. Results: Hypoxia induced profound changes in ECG ST-segment and troponin I leakage in ApoE(-/-)/LDLR-/- mice but not in WT mice. The evaluation of the cardiac proteomic pattern revealed that ApoE(-/-)/LDLR-/- as compared with WT mice were characterised by coordinated increased expression of mitochondrial proteins, including enzymes of fatty acids' and branched-chain amino acids' oxidation, accompanied by decreased expression levels of glycolytic enzymes. These findings correlated with in vivo analysis, revealing a reduction in the entry of glucose and enhanced entry of leucine into the cardiac Krebs cycle, with the cardiac high-energy phosphates pool maintained. These changes were accompanied by the activation of molecular targets controlling mitochondrial metabolism. Ranolazine reversed the oxidative metabolic shift in ApoE(-/-)/LDLR-/- mice and reduced cardiac damage induced by hypoxia. Conclusions: Wesuggest a novelmechanism formyocardial injury in dyslipidaemia that is consequent to an increased reliance on oxidativemetabolismin the heart. The alterations in themetabolic pattern thatwe identified constitute an adaptive mechanism that facilitates maintenance of metabolic equilibrium and cardiac function under normoxia. However, this adaptation could account for myocardial injury even in a mild reduction of oxygen supply. (C) 2020 Elsevier Inc. All rights reserved.

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.

Phage vB_SauP_phiAGO1.3 (phiAGO1.3) is a polyvalent Staphylococcus lytic podovirus with a 17.6-kb genome (Gozdek et al., 2018). It can infect most of the Staphylococcus aureus human isolates of dominant clonal complexes. We show that a major factor contributing to the wide host range of phiAGO1.3 is a lack or sparcity of target sites for certain restriction-modification systems of types I and II in its genome. Phage phiAGO1.3 requires for adsorption β-O-GlcNAcylated cell wall teichoic acid, which is also essential for the expression of methicillin resistance. Under certain conditions an exposure of S. aureus to phiAGO1.3 can lead to the establishment of a mixed population in which the bacteria and phages remain in equilibrium over multiple generations. This is reminiscent of the so called phage carrier state enabling the co-existence of phage-resistant and phage-sensitive cells supporting a continuous growth of the bacterial and phage populations. The stable co-existence of bacteria and phage favors the emergence of phage-resistant variants of the bacterium. All phiAGO1.3-resistant cells isolated from the phage-carrier-state cultures contained a mutation inactivating the two-component regulatory system ArlRS, essential for efficient expression of numerous S. aureus virulence-associated traits. Moreover, the mutants were unaffected in their susceptibility to infection with an unrelated, polyvalent S. aureus phage of the genus Kayvirus. The ability of phiAGO1.3 to establish phage-carrier-state cultures did not preclude its antistaphylococcal activity in vivo in an S. aureus nematode infection model. Taken together our results suggest that phiAGO1.3 could be suitable for the therapeutic application in humans and animals, alone or in cocktails with Kayvirus phages. It might be especially useful in the treatment of infections with the majority of methicillin-resistant S. aureus strains.

Summary The disaccharide trehalose is widely distributed in nature and can serve as a carbon reservoir, a signaling molecule for controlling glucose metabolism and a stress protectant. We demonstrated that in Escherichia coli Δ otsA cells, which are unable to synthesize trehalose, the aggregation of endogenous proteins during the stationary phase was increased in comparison to wild‐type cells. The lack of trehalose synthesis boosted Nε‐lysine acetylation of proteins, which in turn enhanced their hydrophobicity and aggregation. This increased Nε‐lysine acetylation could result from carbon overflow and the accumulation of acetyl phosphate caused by the Δ otsA mutation. These findings provide a better understanding of the previously reported protective functions of trehalose in protein stabilization and the prevention of protein aggregation. Our results indicate that trehalose may participate in proteostasis not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation. We propose that trehalose protects E. coli against carbon stress – the synthesis and storage of trehalose can prevent carbon overflow, which otherwise is manifested by protein acetylation and aggregation.

This study aims the detection of proteins associated with increased resistance of tubers to necrotrophic bacteria Dickeya solani in tetraploid and diploid potato plants. Comparative analysis of differently expressed proteins in tuber tissue of potato cultivars and diploid interspecific hybrids of Solanum, differing in resistance to Dickeya solani, was performed using nano-liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS/MS). Two highly resistant (Bea and Humalda) and three susceptible (Irys, Katahdin, Ulster Supreme) potato cultivars, and the highly resistant (DG 00–270) and the susceptible (DG 08–305) diploid clones, were studied. Proteins were extracted from wounded potato tubers inoculated with bacteria at an early symptomatic phase of infection and from controls, i.e., intact tubers and wounded mock-inoculated tubers. Data are available via ProteomeXchange with identifier PXD013009. Eight constitutive differentially expressed proteins with fold changes ≥1.9 and q-value ≤0.1 between the resistant and susceptible cultivar groups after D. solani infection were selected. Probable inactive patatin-03-Kuras 1 and the proteinase inhibitor PTI exhibited significantly increased protein abundances after bacterial inoculation in both resistant cultivars compared to the susceptible cultivars. In the diploid clones, only metallocarboxypeptidase and metallocarboxypeptidase-like inhibitors exhibited much higher fold changes following pathogenic invasion (274.4- and 368.6-fold, respectively) than after mock inoculation (165.5- and 130.7-fold, respectively). These results show that different proteins indicating significant fold changes between the resistant and susceptible potato cultivars and diploid clones are induced at an early phase of symptomatic D. solani infection.

Seedlings of spring wheat (Triticum aestivum L.) cultivars, Ethos and Zebra, differing in drought tolerance were dehydrated to reach a water saturation deficit (WSD) in leaves ~15, 30, and 50 %.Ethos, the drought tolerant cultivar, dried slower in comparison with Zebra and regrew in 70 % upon rehydration.The effect of dehydration on photosystem II was evaluated by Chl a fluorescence (OJIP transients).The inflection point of double normalized curves (ΔW OJ ) calculated for Ethos was negative for seedlings with 15 % WSD, nearly zero for those with 30 % WSD, and about +0.05 for those with 50 % WSD.In case of Zebra, the 15 % WSD already induced a positive ΔW OJ (+0.05) and 50 % WSD maximized it to +0.10, which is a sign of drought susceptibility.The proteomic studies revealed, that among identified 850 spots, 80 protein spots were differentially expressed during dehydration.The differentially expressed proteins of the drought tolerant cultivar indicated the protection of the photosynthetic apparatus and proteome rebuilding in response to drought.In the drought susceptible cultivar, protection of proteins and membranes and partial scavenging reactive oxygen species appeared.

The quail oviduct (Coturnix c. japonica) is a natural candidate avian bioreactor, while the secretive quail oviduct epithelial cells (QOECs) are potential in vitro producers of recombinant proteins and vaccines. In view of the need for highly performing and transformable cell lines, QOEC may potentially act as an alternative bioreactor platform to the existing ones, for example, to the Chinese hamster ovary. The aim of this work was to characterize QOECs and their response to nucleofection with a nonviral plasmid DNA carrying the human interferon-α 2a gene (hIFNλ2a), in vitro. Primary QOEC cultures from laying quails (10-15 weeks old) were characterized by their proliferation rate, doubling time, and multilineage differentiation. Electroporation to cell nuclei (nucleofection) was used to deliver nonviral plasmid DNA containing a reporter GFP and hIFN under the ovalbumin promoter. The posttransfection analysis included polymerase chain reaction, Western blot analysis, and liquid chromatography coupled to tandem mass spectrometry. QOEC showed a typical epithelial characteristic in a primary 2D monolayer culture system and retained secretive potential up to the first passage. QOEC showed differentiation into osteoblastic lineage after stimulation. The nucleofection mean efficiency was low (2.3%). Differences of up to 10% in the proteomic profiles between nontransfected and transfected QOEC were found, the most important of these were related to the absence of keratins and cell-adhesion proteins in the transfected QOEC. Concluding, with the practical information provided here, QOEC have the potential to serve as an avian secreting cellular platform. QOEC may be further transformed to cell lineage to meet the requirement for a stable, electrocompetent, and transfectable model. The first proteomic comparison of QOEC delivered in this study showed, in the majority, a stable proteome of the nontransfected vs transfected QOEC.

The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81-176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains - ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81–176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains – ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.

BACKGROUND Stilbenes, 1,2-diphenylethen derivatives, including resveratrol and combretastatins, show anticancer features especially against tumor angiogenesis. Fosbretabulin, CA-4, in combination with carboplatin, is in the last stages of clinical tests as an inhibitor of thyroid cancer. The mode of action of these compounds involves suppression of angiogenesis through interfering with tubulin (de)polymerization. OBJECTIVE We have previously synthesized five E-2-hydroxystilbenes and seven dibenzo [b,f]oxepins in Z configuration, with methyl or nitro groups at varied positions. The aim of the present work was to evaluate the anticancer activity and molecular mechanism(s) of action of these compounds. RESULTS Two healthy, EUFA30 and HEK293, and two cancerous, HeLa and U87, cell lines were treated with four newly synthetized stilbenes and seven oxepins. Two of these compounds, JJR5 and JJR6, showed the strongest cytotoxic effect against cancerous cells tested and these two were selected for further investigations. They induced apoptosis with sub-G1 or S cell cycle arrest and PARP cleavage, with no visible activation of caspases 3 and 7. Proteomic differential analysis of stilbene-treated cells led to the identification of proteins involved almost exclusively in cell cycle management, apoptosis, DNA repair and stress response, e.g. oxidative stress. CONCLUSION Among the newly synthesized stilbene derivatives, we selected two as potent anticancer compounds triggering late apoptosis/necrosis in cancerous cells through sub-G1 phase cell cycle arrest. They changed cyclin expression, induced DNA repair mechanisms, enzymes involved in apoptosis and oxidative stress response. Compounds JJR5 and JJR6 can be a base for structure modification(s) to obtain even more active derivatives.

SNF1-related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA-dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA-non-activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S-segment is involved in the regulation of dehydrin subcellular localization in response to stress.

Jay Amin hydroxamic acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA's cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 μM. JAHA's lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction, and autophagy mechanisms. To glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination of differential display-PCR, proteome analysis, and COMET assay techniques and shown some differences in the molecular signature profiles induced by exposure to either HDACis. In particular, in contrast to the more numerous and diversified changes induced by SAHA, JAHA has shown a more selective impact on expression of molecular signatures involved in antioxidant activity and DNA repair. Besides expanding the biological knowledge of the effect exerted by the modifications in compound structures on cell phenotype, the molecular elements put in evidence in our study may provide promising targets for therapeutic interventions on TNBCs.