In a screen for gene copy-number changes in mouse mammary tumors, we identified a tumor with a small 350-kb amplicon from a region that is syntenic to a much larger locus amplified in human cancers at chromosome 11q22. The mouse amplicon contains only one known gene, Yap, encoding the mammalian ortholog of Drosophila Yorkie (Yki), a downstream effector of the Hippo(Hpo)-Salvador(Sav)-Warts(Wts) signaling cascade, recently identified in flies as a critical regulator of cellular proliferation and apoptosis. In nontransformed mammary epithelial cells, overexpression of human YAP induces epithelial-to-mesenchymal transition, suppression of apoptosis, growth factor-independent proliferation, and anchorage-independent growth in soft agar. Together, these observations point to a potential oncogenic role for YAP in 11q22-amplified human cancers, and they suggest that this highly conserved signaling pathway identified in Drosophila regulates both cellular proliferation and apoptosis in mammalian epithelial cells.

https://www.pnas.org/content/pnas/103/33/12405.full.pdf

Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Global Analysis of Protein Phosphorylation in Yeast

Mapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward. We report a simple but effective tool, the D rosophila RNAi Screening Center I ntegrative O rtholog P rediction T ool (DIOPT; http://www.flyrnai.org/diopt
 ), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist
 ). DIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

/pdf/an-integrative-approach-to-ortholog-prediction-for-disease-367diozpqg.pdf

An integrative approach to ortholog prediction for disease-focused and other functional studies

C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.

/pdf/modifiers-of-c9orf72-dipeptide-repeat-toxicity-connect-2np3v0nske.pdf

Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS

Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes, but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitochondria in Saccharomyces cerevisiae, and show that mitochondrial dysfunction in replicatively aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asymmetric divisions of a mother cell, and that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degradation, but rather by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient-sensing pathways. Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential. Overall, our results identify vacuolar pH as a critical regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because the functions of the vacuole are highly conserved throughout evolution, we propose that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.

/pdf/an-early-age-increase-in-vacuolar-ph-limits-mitochondrial-4nwudrq084.pdf

An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast

Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events which occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cell-based assays that monitor changes in cell proliferation, migration, and morphogenesis in MCF-10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.

/pdf/functional-proteomics-approach-to-investigate-the-biological-39ojcrham0.pdf

Functional proteomics approach to investigate the biological activities of cDNAs implicated in breast cancer.

The rapid development of new technologies for the high throughput (HT) study of proteins has increased the demand for comprehensive plasmid clone resources that support protein expression. These clones must be full-length, sequence-verified and in a flexible format. The generation of these resources requires automated pipelines supported by software management systems. Although the availability of clone resources is growing, current collections are either not complete or not fully sequence-verified. We report an automated pipeline, supported by several software applications that enabled the construction of the first comprehensive sequence-verified plasmid clone resource for more than 96% of protein coding sequences of the genome of F. tularensis, a highly virulent human pathogen and the causative agent of tularemia. This clone resource was applied to a HT protein purification pipeline successfully producing recombinant proteins for 72% of the genes. These methods and resources represent significant technological steps towards exploiting the genomic information of F. tularensis in discovery applications.

/pdf/a-full-genomic-sequence-verified-protein-coding-gene-2ylthx5ksl.pdf

A Full-Genomic Sequence-Verified Protein-Coding Gene Collection for Francisella tularensis

The availability of an annotated genome sequence for the yeast Saccharomyces cerevisiae has made possible the proteome-scale study of protein function and protein–protein interactions. These studies rely on availability of cloned open reading frame (ORF) collections that can be used for cell-free or cell-based protein expression. Several yeast ORF collections are available, but their use and data interpretation can be hindered by reliance on now out-of-date annotations, the inflexible presence of N- or C-terminal tags, and/or the unknown presence of mutations introduced during the cloning process. High-throughput biochemical and genetic analyses would benefit from a “gold standard” (fully sequence-verified, high-quality) ORF collection, which allows for high confidence in and reproducibility of experimental results. Here, we describe Yeast FLEXGene, a S. cerevisiae protein-coding clone collection that covers over 5000 predicted protein-coding sequences. The clone set covers 87% of the current S. cerevisiae genome annotation and includes full sequencing of each ORF insert. Availability of this collection makes possible a wide variety of studies from purified proteins to mutation suppression analysis, which should contribute to a global understanding of yeast protein function.

/pdf/approaching-a-complete-repository-of-sequence-verified-4oo85ggbxc.pdf

Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae

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Functional proteomics approach to investigate the biological activities of cDNAs implicated in breast cancer.

A Full-Genomic Sequence-Verified Protein-Coding Gene Collection for Francisella tularensis

Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae