RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways

Identification of nuclear genes affecting 2-Deoxyglucose resistance inSchizosaccharomyces pombe

2-Deoxyglucose (2-DG) is a toxic glucose analog. To identify genes involved in 2-DG toxicity in Schizosaccharomyces pombe, we screened a wild-type overexpression library for genes which render cells 2-DG resistant. A gene we termed odr1, encoding an uncharacterized hydrolase, led to strong resistance and altered invertase expression when overexpressed. We speculate that Odr1 neutralizes the toxic form of 2-DG, similar to the Saccharomyces cerevisiae Dog1 and Dog2 phosphatases which dephosphorylate 2-DG-6-phosphate synthesized by hexokinase. In a complementary approach, we screened a haploid deletion library to identify 2-DG-resistant mutants. This screen identified the genes snf5, ypa1, pas1 and pho7. In liquid medium, deletions of these genes conferred 2-DG resistance preferentially under glucose-repressed conditions. The deletion mutants expressed invertase activity more constitutively than the control strain, indicating defects in the control of glucose repression. No S. cerevisiae orthologs of the pho7 gene is known, and no 2-DG resistance has been reported for any of the deletion mutants of the other genes identified here. Moreover, 2-DG leads to derepressed invertase activity in S. pombe, while in S. cerevisiae it becomes repressed. Taken together, these findings suggest that mechanisms involved in 2-DG resistance differ between budding and fission yeasts

A genetic analysis of nitric oxide-mediated signaling during chronological aging in the yeast

In mammals, NO•, a signaling molecule is implicated in the regulation of vasodilation, neurotransmission and immune response. It is believed that NO• is a signaling molecule also in unicellular organism like yeast and may be involved in the regulation of apoptosis and sporulation. It has been reported that NO• is produced during chronological aging (CA) leading to an increase of the superoxide level, which in turn mediates apoptosis. Since this conclusion was based on indirect measurements of NO• by the Griess reaction, the role of NO• signaling during CA in the yeast remains uncertain. We investigated this issue more precisely using different genetic and biochemical methodologies. We used cells lacking the factors influencing nitrosative stress response like flavohemoglobin metabolizing NO•, S-nitrosoglutathione reductase metabolizing S-nitrosoglutathione and the transcription factor Fzf1p mediating NO• response. We measured the standard parameters describing CA and found an elevation in the superoxide level, percentage of death cells, the level of TUNEL positive cells and a decrease in proliferating potential. These observations showed no significant differences between wild type cells and the disruptants except for a small elevation of the superoxide level in the Δsfa1 mutant. The intracellular NO• level and flavohemoglobin expression decreased rather than increased during CA. Products of general nitrogen metabolism and protein tyrosine nitration were slightly decreased during CA, the magnitude of changes showing no differences between the wild type and the mutant yeast. Altogether, our data indicate that apoptosis during yeast CA is mediated by superoxide signaling rather than NO• signaling

Nitric oxide as a signaling molecule in the fission yeast Schizosaccharomyces pombe

Nitric oxide synthases (NOS) catalyze the synthesis of ubiquitous signaling molecule nitric oxide (NO) which controls numerous biological processes. Using a spectrofluorometric NOS assay, we have measured the rate of total NO production in the crude cell extracts of Schizosaccharomyces pombe. NO production was reduced in the absence of NOS cofactors calmodulin and tetrahydrobiopterin, and a competitive NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) was able to cause a statistically significant inhibition on the rate of total NO production. These results, for the first time, provide evidence that an enzyme with a NOS-like activity may be present in the fission yeast. In order to assess the possible regulatory roles of NO as a signaling molecule in this yeast, using the differential display technique, we screened for NO-responsive genes whose expression decreased upon exposure to l-NAME and increased in response to an NO donor, sodium nitroprusside treatment. Differential expression patterns of byr1, pek1, sid1, and wis1 genes were confirmed by quantitative real-time PCR. The physiological experiments performed based on the functions and molecular interactions of these genes have pointed to the possibility that NO production might be required for sporulation in S. pombe. Taken together, these findings suggest that NO may function as a signaling molecule which can induce both transcriptional and physiological changes in the fission yeast. Hence, these data also imply that S. pombe can be used as a model system for investigating the mechanisms underlying NO-related complex signaling pathways

Isolation and characterization of glucose derepressed invertase mutants from Schizosaccharomyces pombe

We have isolated 14 different Schizosaccharontyces pombe mutants that synthesize invertase enzyme constitutively. Analyses of invertase activities revealed that the degrees of resistance to glucose repression were not similar among different complementation groups. One of the complementation groups appeared to be associated with functional and/or regulatory defects in hexose transport. Another complementation group appeared to be specific for the regulation of the inv1 gene alone, implying that these mutations might be associated with different genes acting on the glucose sensing and signaling pathway. In addition, we found that the wild-type level glucose uptake is essential for the full-level repression of inv1 expression

Association between active pulmonary tuberculosis and circulating microRNAs: a preliminary study from Turkey

Background/aim: Tuberculosis is a public health problem that still remains significant. For prevention, diagnosis, and treatment of tuberculosis more effective novel biomarkers are needed. MicroRNAs can regulate innate and adaptive immune responses, alter host-pathogen interactions, and affect progression of diseases. The relationship between microRNA expression and active pulmonary tuberculosis (APT) has not yet been investigated in the Turkish population. We aimed to test the potential diagnostic value of some microRNAs whose levels were previously reported to be altered in APT patients. Materials and methods: Using two different references (U6 and miR-93), we compared the expression levels of potentially important microRNAs in serum of APT patients with healthy individuals using quantitative polymerase chain reaction (qPCR). Results: miR-144 expression level was down-regulated in APT patients when either U6 or miR-93 was used for normalization. When data was normalized with miR-93, a statistically significant decrease in miR-125b (0.8 fold) and miR-146a (0.7 fold) expression levels were observed, while no differences were detected for U6. The receiver operating characteristic suggested that miR-144 may be a candidate biomarker for discriminating APT patients and controls (p < 0.05) both for U6 and miR-93. Conclusion: These findings suggest that miR-144 can have potential as a biomarker for APT. Using a single reference may be misleading in evaluation of microRNA expression. U6 and miR-93 can be used in combination as references for normalization of serum microRNA expression data