Katayoun Omidi

Recent advances in protein-protein interaction prediction: experimental and computational methods.

Introduction: Proteins within the cell act as part of complex networks, which allow pathways and processes to function. Therefore, understanding how proteins interact is a significant area of current research. Areas covered: This review aims to present an overview of key experimental techniques (yeast two-hybrid, tandem affinity purification and protein microarrays) used to discover protein–protein interactions (PPIs), as well as to briefly discuss certain computational methods for predicting protein interactions based on gene localization, phylogenetic information, 3D structural modeling or primary protein sequence data. Due to the large-scale applicability of primary sequence-based methods, the authors have chosen to focus on this strategy for our review. There is an emphasis on a recent algorithm called Protein Interaction Prediction Engine (PIPE) that can predict global PPIs. The readers will discover recent advances both in the practical determination of protein interaction and the strategies that are available to attempt to anticipate interactions without the time and costs of experimental work. Expert opinion: Global PPI maps can help understand the biology of complex diseases and facilitate the identification of novel drug target sites. This study describes different techniques used for PPI prediction that we believe will significantly impact the development of the field in a new future. We expect to see a growing number of similar techniques capable of large-scale PPI predictions.

Phosphatase complex Pph3/Psy2 is involved in regulation of efficient non-homologous end-joining pathway in the yeast Saccharomyces cerevisiae.

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.

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining.

ABSTRACT The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.

Zinc oxide and silver nanoparticles toxicity in the baker's yeast, Saccharomyces cerevisiae

Engineered nanomaterials (ENMs) are increasingly incorporated into a variety of commercial applications and consumer products; however, ENMs may possess cytotoxic properties due to their small size. This study assessed the effects of two commonly used ENMs, zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs), in the model eukaryote Saccharomyces cerevisiae. A collection of ≈4600 S. cerevisiae deletion mutant strains was used to deduce the genes, whose absence makes S. cerevisiae more prone to the cytotoxic effects of ZnONPs or AgNPs. We demonstrate that S. cerevisiae strains that lack genes involved in transmembrane and membrane transport, cellular ion homeostasis, and cell wall organization or biogenesis exhibited the highest sensitivity to ZnONPs. In contrast, strains that lack genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport exhibited the highest sensitivity to AgNPs. Secondary assays confirmed that ZnONPs affected cell wall function and integrity, whereas AgNPs exposure decreased transcription, reduced endocytosis, and led to a dysfunctional electron transport system. This study supports the use of S. cerevisiae Gene Deletion Array as an effective high-throughput technique to determine cellular targets of ENM toxicity.

The sensitivity of the yeast, Saccharomyces cerevisiae, to acetic acid is influenced by DOM34 and RPL36A

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.

Designing anti-Zika virus peptides derived from predicted human-Zika virus protein-protein interactions

The production of anti-Zika virus (ZIKV) therapeutics has become increasingly important as the propagation of the devastating virus continues largely unchecked. Notably, a causal relationship between ZIKV infection and neurodevelopmental abnormalities has been widely reported, yet a specific mechanism underlying impaired neurological development has not been identified. Here, we report on the design of several synthetic competitive inhibitory peptides against key pathogenic ZIKV proteins through the prediction of protein-protein interactions (PPIs). Often, PPIs between host and viral proteins are crucial for infection and pathogenesis, making them attractive targets for therapeutics. Using two complementary sequence-based PPI prediction tools, we first produced a comprehensive map of predicted human-ZIKV PPIs (involving 209 human protein candidates). We then designed several peptides intended to disrupt the corresponding host-pathogen interactions thereby acting as anti-ZIKV therapeutics. The data generated in this study constitute a foundational resource to aid in the multi-disciplinary effort to combat ZIKV infection, including the design of additional synthetic proteins.

Uncharacterized ORF HUR1 influences the efficiency of non-homologous end-joining repair in Saccharomyces cerevisiae

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.

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples

A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella -specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non- Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

Heavy metal sensitivities of gene deletion strains for ITT1 and RPS1A connect their activities to the expression of URE2, a key gene involved in metal detoxification in yeast

Heavy metal and metalloid contaminations are among the most concerning types of pollutant in the environment. Consequently, it is important to investigate the molecular mechanisms of cellular responses and detoxification pathways for these compounds in living organisms. To date, a number of genes have been linked to the detoxification process. The expression of these genes can be controlled at both transcriptional and translational levels. In baker’s yeast, Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferases. Yeast URE2 encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. The URE2 expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes, ITT1, an inhibitor of translation termination, and RPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain for URE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affect URE2 mRNA expression at the level of translation.