Dissecting Ubiquitylation and DNA Damage Response Pathways in the Yeast Saccharomyces cerevisiae Using a Proteome-Wide Approach.

In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR depends upon various posttranslational modifications; among them, ubiquitylation plays a key regulatory role. Here, we profiled ubiquitylation in response to the DNA alkylating agent methyl methanesulfonate (MMS) in the budding yeast Saccharomyces cerevisiae using quantitative proteomics. To discover new proteins ubiquitylated upon DNA replication stress, we used stable isotope labeling by amino acids in cell culture, followed by an enrichment of ubiquitylated peptides and LC-MS/MS. In total, we identified 1853 ubiquitylated proteins, including 473 proteins that appeared upregulated more than 2-fold in response to MMS treatment. This enabled us to localize 519 ubiquitylation sites potentially regulated upon MMS in 435 proteins. We demonstrated that the overexpression of some of these proteins renders the cells sensitive to MMS. We also assayed the abundance change upon MMS treatment of a selection of yeast nuclear proteins. Several of them were differentially regulated upon MMS treatment. These findings corroborate the important role of ubiquitin-proteasome-mediated degradation in regulating the DDR.

Protein-fragment complementation assays for large-scale analysis of protein-protein interactions.

Protein-protein interactions (PPIs) orchestrate nearly all biological processes. They are also considered attractive drug targets for treating many human diseases, including cancers and neurodegenerative disorders. Protein-fragment complementation assays (PCAs) provide a direct and straightforward way to study PPIs in living cells or multicellular organisms. Importantly, PCAs can be used to detect the interaction of proteins expressed at endogenous levels in their native cellular environment. In this review, we present the principle of PCAs and discuss some of their advantages and limitations. We describe their application in large-scale experiments to investigate PPI networks and to screen or profile PPI targeting compounds.

Protein quality control at the inner nuclear membrane.

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

Transcription Factors and Markers Related to Epithelial-Mesenchymal Transition and Their Role in Resistance to Therapies in Head and Neck Cancers.

Head and neck cancers (HNCs) are heterogeneous and aggressive tumors of the upper aerodigestive tract. Although various histological types exist, the most common is squamous cell carcinoma (HNSCC). The incidence of HNSCC is increasing, making it an important public health concern. Tumor resistance to contemporary treatments, namely, chemo- and radiotherapy, and the recurrence of the primary tumor after its surgical removal cause huge problems for patients. Despite recent improvements in these treatments, the 5-year survival rate is still relatively low. HNSCCs may develop local lymph node metastases and, in the most advanced cases, also distant metastases. A key process associated with tumor progression and metastasis is epithelial-mesenchymal transition (EMT), when poorly motile epithelial tumor cells acquire motile mesenchymal characteristics. These transition cells can invade different adjacent tissues and finally form metastases. EMT is governed by various transcription factors, including the best-characterized TWIST1 and TWIST2, SNAIL, SLUG, ZEB1, and ZEB2. Here, we highlight the current knowledge of the process of EMT in HNSCC and present the main protein markers associated with it. This review focuses on the transcription factors related to EMT and emphasizes their role in the resistance of HNSCC to current chemo- and radiotherapies. Understanding the role of EMT and the precise molecular mechanisms involved in this process may help with the development of novel anti-cancer therapies for this type of tumor.

Analysis of age-related global DNA methylation in chicken.

DNA methylation is an epigenetic modification that plays an important role in the normal development and function of organisms. The level of DNA methylation is species-, tissue-, and organelle-specific, and the methylation pattern is determined during embryogenesis. DNA methylation has also been correlated with age. The aim of this study was to determine the global DNA methylation levels and their correlation with age in the chicken, using a Polish autosexing chicken breed, Polbar. A quantitative technique based on an immunoenzymatic assay was used for global DNA methylation analysis. The results show increased global DNA methylation levels with older Polbar embryos. Global DNA methylation levels decrease with the age of hens in the postembryonic stage. This study expands the current knowledge of the Polbar epigenome and the general knowledge of the function of epigenetic mechanisms in birds.

Estrogen Receptor Signaling in Breast Cancer.

Estrogen receptor (ER) signaling is a critical regulator of cell proliferation, differentiation, and survival in breast cancer (BC) and other hormone-sensitive cancers. In this review, we explore the mechanism of ER-dependent downstream signaling in BC and the role of estrogens as growth factors necessary for cancer invasion and dissemination. The significance of the clinical implications of ER signaling in BC, including the potential of endocrine therapies that target estrogens' synthesis and ER-dependent signal transmission, such as aromatase inhibitors or selective estrogen receptor modulators, is discussed. As a consequence, the challenges associated with the resistance to these therapies resulting from acquired ER mutations and potential strategies to overcome them are the critical point for the new treatment strategies' development.

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

Bimolecular Fluorescence Complementation to Assay the Interactions of Ubiquitylation Enzymes in Living Yeast Cells.

Ubiquitylation is a versatile posttranslational protein modification catalyzed through the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). These enzymes form transient complexes with each other and their modification substrates and determine the nature of the ubiquitin signals attached to their substrates. One challenge in the field of protein ubiquitylation is thus to identify the E2-E3 pairs that function in the cell. In this chapter, we describe the use of bimolecular fluorescence complementation to assay E2-E3 interactions in living cells, using budding yeast as a model organism.