Showing papers by "Michael Snyder published in 2005"

Global analysis of protein phosphorylation in yeast

Abstract: 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.

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection

Abstract: Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome.

Advances in functional protein microarray technology.

Abstract: Numerous innovations in high-throughput protein production and microarray surface technologies have enabled the development of addressable formats for proteins ordered at high spatial density. Protein array implementations have largely focused on antibody arrays for high-throughput protein profiling. However, it is also possible to construct arrays of full-length, functional proteins from a library of expression clones. The advent of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein–protein, protein–nucleic acid, and small molecule interactions. This technology holds great potential for basic molecular biology research, disease marker identification, toxicological reponse profiling and pharmaceutical target screening.

Global changes in STAT target selection and transcription regulation upon interferon treatments

Abstract: The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of the genomic loci proximal to these binding sites introduced new candidate STAT1 and STAT2 target genes, several of which are affiliated with proliferation and apoptosis. The genes on chromosome 22 that exhibited interferon-induced up- or down-regulated expression were determined and correlated with the STAT-binding site information, revealing the potential regulatory effects of STAT1 and STAT2 on their target genes. Importantly, the comparison of STAT1-binding sites upon interferon (IFN)-γ and IFN-α treatments revealed dramatic changes in binding locations between the two treatments. The IFN-α induction revealed nonconserved STAT1 occupancy at IFN-γ-induced sites, as well as novel sites of STAT1 binding not evident in IFN-γ-treated cells. Many of these correlated with binding by STAT2, but others were STAT2 independent, suggesting that multiple mechanisms direct STAT1 binding to its targets under different activation conditions. Overall, our results reveal a wealth of new information regarding IFN/STAT-binding targets and also fundamental insights into mechanisms of regulation of gene expression in different cell states.

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening

Abstract: The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified. Using a combination of peptide library selection, phosphorylation of opitmal peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome chip analysis. We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site

Calixarene derivative as a tool for highly sensitive detection and oriented immobilization of proteins in a microarray format through noncovalent molecular interaction

Abstract: One important factor in fabricating protein microarray is to immobilize proteins without losing their activity on a solid phase. To keep them functional, it is necessary to immobilize proteins in a way that preserve their folded structural integrity. In a previous study, we developed novel Calixarene derivatives for the immobilization of proteins on the surface of a glass slide (1). In this study, we compared the sensitivity and the specificity of the linker molecules with those of five other protein attachment agents on glass slides using a prostate-specific antigen and its antibodies as a model system. The Calixcrown-coated protein chip showed a superior sensitivity and a much lower detection limit than those chips prepared by other methods. When we tested the capability of Calixcrown to immobilize antibody molecules, it appeared that Calixcrown makes arrangement of antibody be more regular with the vertical orientation than the covalent-bond agent. We also observed that the Calixcrown chip could be used for the diagnostic application with clinical samples from prostate cancer and HIV patients. Finally, we applied the Calixcrown chip using an antibody microarray to identify up- or down-regulated proteins in specific tissue and detected several up- or down-regulated proteins from a rat liver by administering toxin. Thus, the Calixcrown chip can be used as a powerful tool with a wide range of applications, including protein-protein interaction, protein-DNA interaction, and an enzyme activity assay.

Applications of DNA tiling arrays to experimental genome annotation and regulatory pathway discovery.

Abstract: Microarrays have become a popular and important technology for surveying global patterns in gene expression and regulation. A number of innovative experiments have extended microarray applications beyond the measurement of mRNA expression levels, in order to uncover aspects of large-scale chromosome function and dynamics. This has been made possible due to the recent development of tiling arrays, where all non-repetitive DNA comprising a chromosome or locus is represented at various sequence resolutions. Since tiling arrays are designed to contain the entire DNA sequence without prior consultation of existing gene annotation, they enable the discovery of novel transcribed sequences and regulatory elements through the unbiased interrogation of genomic loci. The implementation of such methods for the global analysis of large eukaryotic genomes presents significant technical challenges. Nonetheless, tiling arrays are expected to become instrumental for the genome-wide identification and characterization of functional elements. Combined with computational methods to relate these data and map the complex interactions of transcriptional regulators, tiling array experiments can provide insight toward a more comprehensive understanding of fundamental molecular and cellular processes.

Spindle checkpoint maintenance requires Ame1 and Okp1.

Abstract: Kinetochore proteins are required for high fidelity chromosome segregation and as a platform for checkpoint signaling. Ame1 is an essential component of the COMA (Ctf19, Okp1, Mcm21, Ame1) sub-complex of the central kinetochore of budding yeast. In this study, we describe the isolation and characterization of an Ame1 conditional mutant, ame1-4. ame1-4 cells exhibit chromosome segregation defects and Mad2-dependent cell cycle delay similar to okp1-5 cells. However, the viability of ame1-4 cells is markedly reduced relative to wild type and okp1-5 cells after three hours at restrictive temperature. To determine if ame1-4 cells enter anaphase with mis-segregated chromosomes, we monitored the localization of Bub3:VFP as a marker for anaphase onset. ame1-4 cells containing mis-segregated sister chromatids initially accumulate Bub3:VFP at kinetochores, indicating checkpoint activation and a metaphase arrest. Subsequently, Bub3:VFP de-localizes and cells reinitiate DNA duplication and budding without cytokinesis in the presence of un-segregated chromosomes. Overexpression of OKP1 in ame1-4 cells restores ame1-4 protein localization and a stable arrest. Based on our results, we propose that Ame1 and Okp1 are required for a sustained checkpoint arrest in the presence of mis-segregated chromosomes. Our results suggest that checkpoint response might be controlled not only at the level of activation but also via signals that ensure maintenance of the response.

Prospects and Challenges in Proteomics

Abstract: Proteomics is one of the fastest growing areas in areas of research, largely because the global-scale analysis of proteins is expected to yield more direct understanding of function and regulation than analysis of genes. Although significant advances in the comprehensive profiling, functional

A pilot study of transcription unit analysis in rice using oligonucleotide tiling-path microarray.

Abstract: As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models and in identifying novel transcription units that will facilitate rice genome annotation.

Analysis of genomic tiling microarrays for transcript mapping and the identification of transcription factor binding sites

Abstract: The recently developed technology of genomic tiling microarrays, which can be used for genome annotation, has required the development of new methodologies [Royce et.al] for their design and analysis. Genomic tiling arrays use PCR amplicons or short oligonucleotide probes to tile the non-repetitive DNA sequence of a genome in an unbiased fashion for the purposes of detecting novel genomic features. Specifically, they can be used for the identification of novel transcripts, distinguishing between different splice isoforms and for finding transcription factor binding sites using Chromatin-Immunoprecipitation on chip experiments (ChIP-chip).