For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5'-Untranslated region (5'-UTR) of PGM2 mRNA in yeast.

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.

Manganese (Mn) is an important element; yet acute and/or chronic exposure to this metal has been linked to neurotoxicity and neurodegenerative illnesses such as Parkinson's disease and others via an unknown mechanism. To better understand it, we exposed a human neuroblastoma cell model (SH-SY5Y) to two Mn chemical species, MnCl2 and Citrate of Mn(II) (0-2000 µM), followed by a cell viability assay, transcriptomics, and bioinformatics. Even though these cells have been chemically and genetically modified, which may limit the significance of our findings, we discovered that by using RA-differentiated cells instead of undifferentiated SH-SY5Y cell line, both chemical species induce a similar toxicity, potentially governed by disruption of protein metabolism, with some differences. The MnCl2 altered amino acid metabolism, which affects RNA metabolism and protein synthesis. Citrate of Mn(II), however, inhibited the E3 ubiquitin ligases-target protein degradation pathway, which can lead to the buildup of damaged/unfolded proteins, consistent with histone modification. Finally, we discovered that Mn(II)-induced cytotoxicity in RA-SH-SY5Y cells shared 84 percent of the pathways involved in neurodegenerative diseases.

DNA repair defects are common in tumour cells and can lead to misrepair of double-strand breaks (DSBs), posing a significant challenge to cellular integrity. The overall mechanisms of DSB have been known for decades. However, the list of the genes that affect the efficiency of DSB repair continues to grow. Additional factors that play a role in DSB repair pathways have yet to be identified. In this study, we present a computational approach to identify novel gene functions that are involved in DNA damage repair in Saccharomyces cerevisiae. Among the primary candidates, GAL7, YMR130W, and YHI9 were selected for further analysis since they had not previously been identified as being active in DNA repair pathways. Originally, GAL7 was linked to galactose metabolism. YHI9 and YMR130W encode proteins of unknown functions. Laboratory testing of deletion strains gal7Δ, ymr130wΔ, and yhi9Δ implicated all 3 genes in Homologous Recombination (HR) and/or Non-Homologous End Joining (NHEJ) repair pathways, and enhanced sensitivity to DNA damage-inducing drugs suggested involvement in the broader DNA damage repair machinery. A subsequent genetic interaction analysis revealed interconnections of these three genes, most strikingly through SIR2, SIR3 and SIR4 that are involved in chromatin regulation and DNA damage repair network.

One of the main mechanisms for double stranded DNA break (DSB) repair is through the non-homologous end-joining (NHEJ) pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.

Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ.

The presence of acetic acid during industrial alcohol fermentation reduces the yield of fermentation by imposing additional stress on the yeast cells. The biology of cellular responses to stress has been a subject of vigorous investigations. Although much has been learned, details of some of these responses remain poorly understood. Members of heat shock chaperone HSP proteins have been linked to acetic acid and heat shock stress responses in yeast. Both acetic acid and heat shock have been identified to trigger different cellular responses including reduction of global protein synthesis and induction of programmed cell death. Yeast HSC82 and HSP82 code for two important heat shock proteins that together account for 1-2% of total cellular proteins. Both proteins have been linked to responses to acetic acid and heat shock. In contrast to the overall rate of protein synthesis which is reduced, the expression of HSC82 and HSP82 is induced in response to acetic acid stress. In the current study we identified two yeast genes DOM34 and RPL36A that are linked to acetic acid and heat shock sensitivity. We investigated the influence of these genes on the expression of HSP proteins. Our observations suggest that Dom34 and RPL36A influence translation in a CAP-independent manner.

Interest in the evolution of protein-protein and genetic interaction networks has been rising in recent years, but the lack of large-scale high quality comparative datasets has acted as a barrier. Here, we carried out a comparative analysis of computationally predicted protein-protein interaction (PPI) networks from five closely related yeast species. We used the Protein-protein Interaction Prediction Engine (PIPE), which uses a database of known interactions to make sequence-based PPI predictions, to generate high quality predicted interactomes. Simulated proteomes and corresponding PPI networks were used to provide null expectations for the extent and nature of PPI network evolution. We found strong evidence for conservation of PPIs, with lower than expected levels of change in PPIs for about a quarter of the proteome. Furthermore, we found that changes in predicted PPI networks are poorly predicted by sequence divergence. Our analyses identified a number of functional classes experiencing fewer PPI changes than expected, suggestive of purifying selection on PPIs. Our results demonstrate the added benefit of considering predicted PPI networks when studying the evolution of closely related organisms.

Non-Homologous End Joining (NHEJ) is a highly conserved pathway that repairs Double-Strand Breaks (DSBs) within DNA. Here we show that the deletion of yeast uncharacterized ORF HUR1, Hydroxyurea Resistance1 affects the efficiency of NHEJ. Our findings are supported by Protein-Protein Interaction (PPI), genetic interaction and drug sensitivity analyses. To assess the activity of HUR1 in DSB repair, we deleted its non-overlapping region with PMR1, referred to as HUR1-A. We observed that similar to deletion of TPK1 and NEJ1, and unlike YKU70 (important for NHEJ of DNA with overhang and not blunt end), deletion of HUR1-A reduced the efficiency of NHEJ in both overhang and blunt end plasmid repair assays. Similarly, a chromosomal repair assay showed a reduction for repair efficiency when HUR1-A was deleted. In agreement with a functional connection for Hur1p with Tpk1p and NEJ1p, double mutant strains Δhur1-A/Δtpk1, and Δhur1-A/Δnej1 showed the same reduction in the efficiency of plasmid repair, compared to both single deletion strains. Also, using a Homologous Recombination (HR) specific plasmid-based DSB repair assay we observed that deletion of HUR1-A influenced the efficiency of HR repair, suggesting that HUR1 might also play additional roles in other DNA repair pathways.

Manganese (Mn) is an essential element, but in humans, chronic and/or acute exposure to this metal can lead to neurotoxicity and neurodegenerative disorders including Parkinsonism and Parkinson's Disease by unclear mechanisms. To better understand the effects that exposure to Mn2+ exert on eukaryotic cell biology, we exposed a non-essential deletion library of the yeast Saccharomyces cerevisiae to a sub-inhibitory concentration of Mn2+ followed by targeted functional analyses of the positive hits. This screen produced a set of 43 sensitive deletion mutants that were enriched for genes associated with protein biosynthesis. Our follow-up investigations demonstrated that Mn reduced total rRNA levels in a dose-dependent manner and decreased expression of a β-galactosidase reporter gene. This was subsequently supported by analysis of ribosome profiles that suggested Mn-induced toxicity was associated with a reduction in formation of active ribosomes on the mRNAs. Altogether, these findings contribute to the current understanding of the mechanism of Mn-triggered cytotoxicity. Lastly, using the Comparative Toxicogenomic Database, we revealed that Mn shared certain similarities in toxicological mechanisms with neurodegenerative disorders including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's and Huntington's diseases.

Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.