Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, ...we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3L
G204A
or BNIP3L
G208V
, led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3L
S212E
leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression.
Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A
1
; BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl
2
: cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
In the last two decades, accumulating evidence pointed to the importance of autophagy in various human diseases. As an essential evolutionary catabolic process of cytoplasmatic component digestion, ...it is generally believed that modulating autophagic activity, through targeting specific regulatory actors in the core autophagy machinery, may impact disease processes. Both autophagy upregulation and downregulation have been found in cancers, suggesting its dual oncogenic and tumor suppressor properties during malignant transformation. Identification of the key autophagy targets is essential for the development of new therapeutic agents. Despite this great potential, no therapies are currently available that specifically focus on autophagy modulation. Although drugs like rapamycin, chloroquine, hydroxychloroquine, and others act as autophagy modulators, they were not originally developed for this purpose. Thus, autophagy may represent a new and promising pharmacologic target for future drug development and therapeutic applications in human diseases. Here, we summarize our current knowledge in regard to the interplay between autophagy and malignancy in the most significant tumor types: pancreatic, breast, hepatocellular, colorectal, and lung cancer, which have been studied in respect to autophagy manipulation as a promising therapeutic strategy. Finally, we present an overview of the most recent advances in therapeutic strategies involving autophagy modulators in cancer.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the ...Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Here, we report a comparative study of the phytochemical profile and the biological activity of two onion extracts, namely
L. and
×
(Clementi ex Visiani 1842), members of the family Amaryllidaceae. ...The identification of flavonoids and anthocyanins, and their individual quantities, was determined by high-performance liquid chromatography (HPLC). The potency of both extracts to scavenge free radicals was determined by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) methods. The DNA protective role was further tested by the single-cell gel electrophoresis (COMET) assay and by Fenton's reagent causing double-strand breaks on the closed circular high copy pUC19 plasmid isolated from
. In the presence of both extracts, a significant decrease in DNA damage was observed, which indicates a protective role of
and
×
on DNA strand breaks. Additionally, cytotoxicity was tested on glioblastoma and breast cancer cell lines. The results showed that both extracts had antiproliferative effects, but the most prominent decrease in cellular growth was observed in glioblastoma cells.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
This study is aimed to better understand the bactericidal mode of action of silver nanoparticles. Here we present the production and characterization of laser-synthesized silver nanoparticles along ...with growth curves of bacteria treated at sub-minimal and minimal inhibitory concentrations, obtained by optical density measurements. The main effect of the treatment is the increase of the bacterial apparent lag time, which is very well described by the novel growth model as well as the entire growth curves for different concentrations. The main assumption of the model is that the treated bacteria uptake the nanoparticles and inactivate, which results in the decrease of both the nanoparticles and the bacteria concentrations. The lag assumes infinitive value for the minimal inhibitory concentration treatment. This apparent lag phase is not postponed bacterial growth. It is a dynamic state in which the bacterial growth and death rates are close in value. Our results strongly suggest that the predominant mode of antibacterial action of silver nanoparticles is the penetration inside the membrane.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Quaternary ammonium compounds (QACs) are among the most effective antimicrobial agents that have been used for more than a century. However, due to the growing trend of bacterial resistance and high ...toxicity of QACs, research in this field remains a pressing matter. Recent studies of the structure-activity relationship suggest that the introduction of the amide functional group into QAC structures results in soft variants that retain their antimicrobial properties while opening the possibility of fine-tuned activity regulation. Here, we report the synthesis and structure-function study of three structurally distinct series of naturally derived soft QACs. The obtained 3-amidoquinuclidine QACs showed a broad range of antibacterial activities related to the hydrophobic-hydrophilic balance of the QAC structures. All three series yielded candidates with minimal inhibitory concentrations (MIC) in the single-digit μM range. Time-resolved growth analysis revealed subtle differences in the antibacterial activity of the selected candidates. The versatile MIC values were recorded in different nutrient media, suggesting that the media composition may have a dramatic impact on the antibacterial potential. The new QACs were found to have excellent potential to suppress bacterial biofilm formation while exhibiting low ability to induce bacterial resistance. In addition, the selected candidates were found to be less toxic than commercially available QACs and proved to be potential substrates for protease degradation. These data suggest that 3-amidoquinuclidine QACs could be considered as novel antimicrobial agents that pose a low threat to ecosystems and human health.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Quaternary ammonium compounds (QACs) are amphiphilic molecules displaying a broad-spectrum of antibacterial activity. QACs are commonly used antiseptics in industrial, home and hospital settings. ...Given the emergence of the QAC-resistant bacteria, there is an urgent need to design new QACs with good antimicrobial activity, able to escape the host resistance mechanism. Therefore, a series of QACs derived from quinuclidine-3-ol and an alkyl chain of variable length (QOH-C3 to -C14), was designed and synthesized. The antimicrobial potential of the new monoquaternary QACs was surveyed against seventeen strains of emerging food spoilage and pathogenic microorganisms, including clinical multidrug-resistant ESKAPE isolates. The QOH-C14 proved to have the strongest antimicrobial activity. It was highly active against all pathogens tested, particularly against the Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.06 to 3.9 μg/mL, and fungi exerting the MIC90 between 0.12 and 3.9 μg/mL. The potency of QOH-C14, confirmed that alkyl chains are an important part of the structure with their lengths playing a critical role in bioactivity of these compounds. The atomic force microscopy images show the disruption of a cell membrane upon the treatment with QOH-C14. These results were additionally confirmed by flow cytometry and fluorescence microscopy. A relatively low toxicity toward healthy human cells underline that QOH-C14 has a potential as new QAC antimicrobial candidate.
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•A series of QACs based on naturally derived quinuclidine was synthesized and evaluated for antimicrobial activity.•Addition of alkyl chains longer than 10 carbon atoms dramatically affect antimicrobial potential of new compounds.•Derivative containing 14 carbon atoms in a side chain, QOH-C14 had the best antimicrobial potential.•Compound QOH-C14 is equally effective as a control, but slower acting in a first few hours of exposure.•The compound is targeting cell membrane and has lower toxicity toward healthy human cell lines than a control.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In plant tissues, enzymes implicated in the lipoxygenase (LOX) pathway are responsible for the hydroperoxydation of polyunsaturated fatty acids, ultimately leading to the production of small chemical ...species involved in several physiological processes. During industrial olive oil production, these enzymes are activated upon crushing and grinding of olive fruit tissue, subsequently leading to the synthesis of volatile compounds responsible for the positive aroma and flavor of the oil. An investigation of LOX activity during olive fruit ripening and malaxation could assist in the production of oils with favorable aroma and taste. Therefore, a reliable method for olive LOX purification is crucial. Here we report a critical review of six LOX extraction protocols, two of which have shown minimum enzyme activity, possibly leading to misconceptions in the interpretation of experimental data. Future research concerning olive LOX should employ extraction methods that preserve enzyme activity.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Species that belong to the genus
have been widely used for human food and traditional medicine. Their beneficial health effects, as well as the specific aroma, are associated with their bioactive ...chemical compounds, such as sulfur compounds and flavonoids. Gas chromatography and mass spectrometry (GC-MS) and reverse-phase high-performance liquid chromatography (reverse-phase HPLC) were used to identify organosulfur and amino acid content of triploid hybrid onion,
Clement ex Visiani, 1842, and common onion,
L.
extracts were tested for their antiproliferative activity in three human cancer cell lines (HeLa, HCT116, and U2OS). DNA fragmentation and DAPI staining analysis were performed on HeLa cells to evaluate the effect of extracts on DNA damage and cell morphology. The mRNA expression of p53, Bax, and Caspase-3 genes involved in apoptosis were analyzed by real-time PCR. Using GC-MS, 27 compounds were found in two
species headspaces. Differences were noted among the main compound abundance in the headspace (although the major thiols and disulfides were qualitatively identic in both
species) and dipropyl disulfide, diisopropyl trisulfide, and (Z)-prop-1-enyl propyl trisulfide were predominant sulfides. Identification of amino acids and their quantities were determined by reverse-phase HPLC. Most abundant amino acids in both onions were arginine (Arg) and glutamic acid (Glu). The results of cytotoxicity testing confirmed antiproliferative effects of both species. The DNA fragmentation assay, DAPI staining and real time PCR analysis confirmed that
and
extracts induced apoptosis in HeLa cells. This study presents the evidence for possible therapeutic use of
and
extracts against human cervical carcinoma cell line.
Quaternary ammonium compounds (QACs) have a long-known application as antiseptics and disinfectants applied in various industries such as pharmaceutical, agricultural and food industry. Given the ...alarming number of QACs resistant bacteria, there is an urgent need to develop new QACs with broad-spectrum antimicrobial activities and low tendency to trigger bacterial resistance. One recently proposed approach to develop new QACs is based on quaternization of natural products, which proved to be successful. Quinuclidine is an interesting natural precursor find to be a part of the structure of biologically active cinchona alkaloids. In addition to the well-established medicinal and pharmaceutical potential of 3-substituted quinuclidines, QACs based on 3-substituted quinuclidines, have only recently been shown to exhibit a significant antimicrobial activity. Most importantly, these compounds exhibit low toxicity toward normal human cell lines, which opens up a new chapter in the QACs field ensuring further investigation of possible therapeutic application of 3-substituted quinuclidine based QACs.
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