Frentizole derivatives with mTOR inhibiting and senomorphic properties.

Frentizole is immunosuppressive drug with low acute toxicity and lifespan-prolonging effect. Recently, frentizole´s potential to disrupt toxic amyloid ß (Aß) - Aß-binding alcohol dehydrogenase (ABAD) interaction in mitochondria in Alzheimer´s brains has been revealed. Another broadly studied drug with anti-aging and immunosuppressive properties is an mTOR inhibitor - rapamycin. Since we do not yet precisely know what is behind the lifespan-prolonging effect of rapamycin and frentizole, whether it is the ability to inhibit the mTOR signaling pathway, reduction in mitochondrial toxicity, immunosuppressive effect, or a combination of all of them, we have decided within our previous work to dock the entire in-house library of almost 240 Aß-ABAD modulators into the FKBP-rapamycin-binding (FRB) domain of mTOR in order to interlink mTOR-centric and mitochondrial free radical-centric theories of aging and thus to increase the chances of success. Based on the results of the docking study, molecular dynamic simulation and MM-PBSA calculations, we have selected nine frentizole-like compounds (1 - 9). Subsequently, we have determined their real physical-chemical properties (logP, logD, pKa and solubility in water and buffer), cytotoxic/cytostatic, mTOR inhibitory, and in vitro anti-senescence (senolytic and senomorphic) effects. Finally, the three best candidates (4, 8, and 9) have been forwarded for in vivo safety studies to assess their acute toxicity and pharmacokinetic properties. Based on obtained results, only compound 4 demonstrated the best results within in vitro testing, the ability to cross the blood-brain barrier and the lowest acute toxicity (LD50 in male mice 559 mg/kg; LD50 in female mice 575 mg/kg).

Discovery of small molecule mechanistic target of rapamycin inhibitors as anti-aging and anti-cancer therapeutics.

To date, the most studied drug in anti-aging research is the mTOR inhibitor - rapamycin. Despite its almost perfect anti-aging profile, rapamycin exerts one significant limitation - inappropriate physicochemical properties. Therefore, we have decided to utilize virtual high-throughput screening and fragment-based design in search of novel mTOR inhibiting scaffolds with suitable physicochemical parameters. Seven lead compounds were selected from the list of obtained hits that were commercially available (4, 5, and 7) or their synthesis was feasible (1, 2, 3, and 6) and evaluated in vitro and subsequently in vivo. Of all these substances, only compound 3 demonstrated a significant cytotoxic, senolytic, and senomorphic effect on normal and cancerous cells. Further, it has been confirmed that compound 3 is a direct mTORC1 inhibitor. Last but not least, compound 3 was found to exhibit anti-SASP activity concurrently being relatively safe within the test of in vivo tolerability. All these outstanding results highlight compound 3 as a scaffold worthy of further investigation.

Synergism of BCL-2 family inhibitors facilitates selective elimination of senescent cells.

Accumulation of senescent cells in tissues with advancing age participates in the pathogenesis of several human age-associated diseases. Specific senescent secretome, the resistance of senescent cells to apoptotic stimuli, and lack of immune system response contribute to the accumulation of senescent cells and their adverse effects in tissues. Inhibition of antiapoptotic machinery, augmented in senescent cells, by BCL-2 protein family inhibitors represents a promising approach to eliminate senescent cells from tissues. This study aimed to explore synergistic and selective senolytic effects of anti-apoptotic BCL-2 family targeting compounds, particularly BH3 mimetics. Using human non-transformed cells RPE-1, BJ, and MRC-5 brought to ionizing radiation-, oncogene-, drug-induced and replicative senescence, we found synergy in combining MCL-1 selective inhibitors with other BH3 mimetics. In an attempt to uncover the mechanism of such synergy, we revealed that the surviving subpopulation of cells resistant to individually applied ABT-737/ABT-263, MIK665, ABT-199, and S63845 BCL-2 family inhibitors showed elevated MCL-1 compared to untreated control cells indicating the presence of a subset of cells expressing high MCL-1 levels and, therefore, resistant to BCL-2 inhibitors within the original population of senescent cells. Overall, we found that combining BCL-2 inhibitors can be beneficial for eliminating senescent cells, thereby enabling use of lower, potentially less toxic, doses of drugs compared to monotherapy, thereby overcoming the resistance of the subpopulation of senescent cells to monotherapy.

Phospho-SIM and exon8b of PML protein regulate formation of doxorubicin-induced rDNA-PML compartment.

Repetitive sequences are among the most unstable regions in the eukaryotic genome and defects in their maintenance correlate with premature aging and cancer development. Promyelocytic leukemia protein (PML) induces accumulation of proteins at distinct nuclear sites, thereby affecting a plethora of processes including DNA repair or maintenance of telomeres. Doxorubicin, the broadly used chemotherapeutic compound, induces formation of PML-nucleolar associations (PNAs). Nevertheless, molecular factors affecting formation of PNAs are still largely unknown. Here we show that PNAs can accumulate ribosomal DNA (rDNA) and, after restoration of RNA polymerase I activity, these structures transfer a fraction of rDNA outside the nucleolus. Mutagenesis of PML isoforms revealed that this process depends on the SUMO-interacting motif and adjacent serine-rich region, and is enhanced by exon8b present exclusively in PML IV isoform. Moreover, we demonstrate that PNAs formation is also regulated by p14ARF/p53 tumor suppressors and casein kinase 2. Our data elucidate how PML nucleolar compartment is assembled, bring the first evidence of PML interacting with rDNA, and show the PML-dependent translocation of rDNA away from the nucleolus.

Dynamic PML protein nucleolar associations with persistent DNA damage lesions in response to nucleolar stress and senescence-inducing stimuli.

Diverse stress insults trigger interactions of PML with nucleolus, however, the function of these PML nucleolar associations (PNAs) remains unclear. Here we show that during induction of DNA damage-induced senescence in human non-cancerous cells, PML accumulates at the nucleolar periphery simultaneously with inactivation of RNA polymerase I (RNAP I) and nucleolar segregation. Using time-lapse and high-resolution microscopy, we followed the genesis, structural transitions and destiny of PNAs to show that: 1) the dynamic structural changes of the PML-nucleolar interaction are tightly associated with inactivation and reactivation of RNAP I-mediated transcription, respectively; 2) the PML-nucleolar compartment develops sequentially under stress and, upon stress termination, it culminates in either of two fates: disappearance or persistence; 3) all PNAs stages can associate with DNA damage markers; 4) the persistent, commonly long-lasting PML multi-protein nucleolar structures (PML-NDS) associate with markers of DNA damage, indicating a role of PNAs in persistent DNA damage response characteristic for senescent cells. Given the emerging evidence implicating PML in homologous recombination-directed DNA repair, we propose that PNAs contribute to sequestration and faithful repair of the highly unstable ribosomal DNA repeats, a fundamental process to maintain a precise balance between DNA repair mechanisms, with implications for genomic integrity and aging.

Highly hydrophilic cationic gold nanorods stabilized by novel quaternary ammonium surfactant with negligible cytotoxicity.

The photothermal cancer therapy using cationic gold nanorods (GNRs) stabilized by quaternary ammonium salts (QAS) have a great potential to enhance conventional cancer treatment as it promises the effective eradication of cancer cells including cells resistant to radio- and chemo-therapy and the stimulation of anti-tumor immune response. However, as the cytotoxicity of the conventional alkanethiol-QAS compounds limits their utility in medicine, here we developed GNRs modified by novel highly hydrophilic cationic surfactant composed of the quaternary ammonium group and ethylene glycol chain N,N,N-trimethyl-3,6,9,12,15-pentaoxaheptadecyl-17-sulfanyl-1-ammonium bromide (POSAB) showing insignificant cytotoxicity in the free state. Surface modification of GNRs by POSAB allowed to prepare nanoparticles with good stability in water, high cellular uptake and localization in lysosomes that are a promising alternative to alkanethiol-stabilized GNRs especially for biomedical applications.

Induction, regulation and roles of neural adhesion molecule L1CAM in cellular senescence.

Aging involves tissue accumulation of senescent cells (SC) whose elimination through senolytic approaches may evoke organismal rejuvenation. SC also contribute to aging-associated pathologies including cancer, hence it is imperative to better identify and target SC. Here, we aimed to identify new cell-surface proteins differentially expressed on human SC. Besides previously reported proteins enriched on SC, we identified 78 proteins enriched and 73 proteins underrepresented in replicatively senescent BJ fibroblasts, including L1CAM, whose expression is normally restricted to the neural system and kidneys. L1CAM was: 1) induced in premature forms of cellular senescence triggered chemically and by gamma-radiation, but not in Ras-induced senescence; 2) induced upon inhibition of cyclin-dependent kinases by p16INK4a; 3) induced by TGFbeta and suppressed by RAS/MAPK(Erk) signaling (the latter explaining the lack of L1CAM induction in RAS-induced senescence); and 4) induced upon downregulation of growth-associated gene ANT2, growth in low-glucose medium or inhibition of the mevalonate pathway. These data indicate that L1CAM is controlled by a number of cell growth- and metabolism-related pathways during SC development. Functionally, SC with enhanced surface L1CAM showed increased adhesion to extracellular matrix and migrated faster. Our results provide mechanistic insights into senescence of human cells, with implications for future senolytic strategies.

Interferon gamma/NADPH oxidase defense system in immunity and cancer.

As a part of cellular pathogen defense, IFNγ triggers induction of NADPH oxidase NOX2, which produces superoxide into phagosomes of immune cells. Recent data show that a similar mechanism can also operate in IFNγ-mediated anticancer control. IFNγ is capable of inducing expression of constitutively active NADPH oxidase NOX4 in tumor cells leading to generation of reactive oxygen species (ROS) damaging DNA, activation of DNA damage response and cell cycle arrest/premature cellular senescence.

New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes.

Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells.

Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping "patchy" pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution.

The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span.

Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth.

Vps factors are required for efficient transcription elongation in budding yeast.

There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin.

Nuclear import of chromatin remodeler Isw1 is mediated by atypical bipartite cNLS and classical import pathway.

The protein Isw1 of Saccharomyces cerevisiae is an imitation-switch chromatin-remodeling factor. We studied the mechanisms of its nuclear import and found that the nuclear localization signal (NLS) mediating the transport of Isw1 into the nucleus is located at the end of the C-terminus of the protein (aa1079-1105). We show that it is an atypical bipartite signal with an unconventional linker of 19 aa (KRIR X(19) KKAK) and the only nuclear targeting signal within the Isw1 molecule. The efficiency of Isw1 nuclear import was found to be modulated by changes to the amino acid composition in the vicinity of the KRIR motif, but not by the linker length. Live-cell imaging of various karyopherin mutants and in vitro binding assays of Isw1NLS to importin-α revealed that the nuclear translocation of Isw1 is mediated by the classical import pathway. Analogous motifs to Isw1NLS are highly conserved in Isw1 homologues of other yeast species, and putative bipartite cNLS were identified in silico at the end of the C-termini of imitation switch (ISWI) proteins from higher eukaryotes. We suggest that the C-termini of the ISWI family proteins play an important role in their nuclear import.

Robust heat shock induces eIF2alpha-phosphorylation-independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast, Saccharomyces cerevisiae.

Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2alpha/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Deltalsm4DeltaC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2alpha became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2alpha. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.

Deregulation of DSE1 gene expression results in aberrant budding within the birth scar and cell wall integrity pathway activation in Saccharomyces cerevisiae.

Strains of Saccharomyces cerevisiae lacking Isw2, the catalytic subunit of the Isw2 chromatin remodeling complex, show the mating type-independent activation of the cell wall integrity (CWI) signaling pathway. Since the CWI pathway activation usually reflects cell wall defects, we searched for the cell wall-related genes changed in expression. The genes DSE1, CTS1, and CHS1 were upregulated as a result of the absence of Isw2, according to previously published gene expression profiles (I. Frydlova, M. Basler, P. Vasicova, I. Malcova, and J. Hasek, Curr. Genet. 52:87-95, 2007). Western blot analyses of double deletion mutants, however, did not indicate the contribution of the chitin metabolism-related genes CTS1 and CHS1 to the CWI pathway activation. Nevertheless, the deletion of the DSE1 gene encoding a daughter cell-specific protein with unknown function suppressed CWI pathway activation in isw2Delta cells. In addition, the deletion of DSE1 also abolished the budding-within-the-birth-scar phenotype of isw2Delta cells. The plasmid-driven overexpression proved that the deregulation of Dse1 synthesis was also responsible for CWI pathway activation and manifestation of the budding-within-the-birth-scar phenotype in wild-type cells. The overproduced Dse1-green fluorescent protein localized to both sides of the septum and persisted in unbudded cells. Although the exact cellular role of this daughter cell-specific protein has to be elucidated, our data point to the involvement of Dse1 in bud site selection in haploid cells.

Special type of pheromone-induced invasive growth in Saccharomyces cerevisiae.

The ability to invade a solid substrate is an important phenomenon due to its connection with pathogenic activity of fungi. We report here on invasion displayed by MATalpha cells of Saccharomyces cerevisiae lacking Isw2p, a subunit of the ISW2 chromatin remodelling complex. We found that on minimal medium, where the isw2Delta MATalpha mutant is not invasive, additional absence of another ISW2 complex subunit, Dls1p or Dpb4p, promoted invasion. Our microarray data showed that derepression of MAT a-specific genes caused by absence of Isw2p is very low. Their expression is increased only by the autocrine activation of the mating pathway. Invasion of isw2Delta MATalpha cells thus resembles the pheromone-induced invasion, including dependence on Fig2p. We show here that another pheromone-induced protein, mating agglutinin Aga1p, can play a role in the agar adhesion necessary for invasion. In contrast with MAT a-cells invading agar under low alpha-pheromone concentration, the invasive growth of isw2Delta cells specifically requires Fus3 kinase. Its function in the invasion of isw2Delta MATalpha cells cannot be completely substituted by Kss1 kinase, which plays a basic role in invasive growth signalling. We suggest that partial dependence of the isw2Delta MATalpha invasion on Fus3p and Aga1p corresponds to a weaker pheromone response of this mutant.