Gregory A. Michaud

Global analysis of protein phosphorylation in yeast

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.

Analyzing antibody specificity with whole proteome microarrays

Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins. Furthermore, new applications such as antibody arrays and monoclonal antibody therapeutics have increased the demand for more specific antibodies to reduce cross-reactivity and side effects. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity, because it allows the simultaneous screening of thousands of proteins for possible cross-reactivity. As an initial test of this approach, we screened 11 polyclonal and monoclonal antibodies to ∼5,000 different yeast proteins deposited on a glass slide and found that, in addition to recognizing their cognate proteins, the antibodies cross-reacted with other yeast proteins to varying degrees. Some of the interactions of the antibodies with noncognate proteins could be deduced by alignment of the primary amino acid sequences of the antigens and cross-reactive proteins; however, these interactions could not be predicted a priori. Our findings show that proteome array technology has potential to improve antibody design and selection for applications in both medicine and research.

SMN2 splice modulators enhance U1–pre-mRNA association and rescue SMA mice

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5 splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

A Critical Role for Cortactin Phosphorylation by Abl-Family Kinases in PDGF-Induced Dorsal-Wave Formation

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.

Comparative analysis of Saccharomyces cerevisiae WW domains and their interacting proteins

BackgroundThe WW domain is found in a large number of eukaryotic proteins implicated in a variety of cellular processes. WW domains bind proline-rich protein and peptide ligands, but the protein interaction partners of many WW domain-containing proteins in Saccharomyces cerevisiae are largely unknown.ResultsWe used protein microarray technology to generate a protein interaction map for 12 of the 13 WW domains present in proteins of the yeast S. cerevisiae. We observed 587 interactions between these 12 domains and 207 proteins, most of which have not previously been described. We analyzed the representation of functional annotations within the network, identifying enrichments for proteins with peroxisomal localization, as well as for proteins involved in protein turnover and cofactor biosynthesis. We compared orthologs of the interacting proteins to identify conserved motifs known to mediate WW domain interactions, and found substantial evidence for the structural conservation of such binding motifs throughout the yeast lineages. The comparative approach also revealed that several of the WW domain-containing proteins themselves have evolutionarily conserved WW domain binding sites, suggesting a functional role for inter- or intramolecular association between proteins that harbor WW domains. On the basis of these results, we propose a model for the tuning of interactions between WW domains and their protein interaction partners.ConclusionProtein microarrays provide an appealing alternative to existing techniques for the construction of protein interaction networks. Here we built a network composed of WW domain-protein interactions that illuminates novel features of WW domain-containing proteins and their protein interaction partners.

Biomarker discovery using protein microarray technology platforms: antibody-antigen complex profiling

Protein microarrays represent an important new tool in proteomic systems biology. This review focuses on the contributions of protein microarrays to the discovery of novel disease biomarkers through antibody-based assays. Of particular interest is the use of protein microarrays for immune response profiling, through which a disease-specific antibody repertoire may be defined. The antigens and antibodies revealed by these studies are useful for clinical assay development, with enormous potential to aid in diagnosis, prognosis, disease staging and treatment selection. The discovery and characterization of novel biomarkers specifically tailored to disease type and stage are expected to enable personalized medicine by facilitating preventative medicine, predictive diagnostics and individualized curative therapies.

Identification of Small Molecule Targets on Functional Protein Microarrays

Small molecules, such as metabolites and hormones, interact with proteins to regulate numerous biological pathways, which are often aberrant in disease. Small molecule drugs have been successfully exploited to specifically perturb such processes and thereby, decrease and even eliminate disease progression. Although there are compelling reasons to fully characterize interactions of small molecules with all proteins from an organism for which an intended drug regimen is planned, currently available technologies are not yet up to this task. High-content functional protein microarrays, containing hundreds to thousands of proteins, are new tools that show great potential for meeting this need. In this chapter, we review examples and methods for profiling small molecules on high-content functional protein arrays and discuss considerations for troubleshooting.

Protein Microarray-Based Screening of Antibody Specificity

The increased use of antibodies as therapeutics, as well as the growing demand for large numbers of antibodies for high-throughput protein analyses, has been accompanied by a need for more specific antibodies. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity since it allows the simultaneous screening of thousands of proteins in relatively normalized quantities. Indeed, the use of a yeast proleome array to profile the specificity of several antibodies directed against yeast proteins has recently been described. In this chapter, we present a detailed description of the methods used to probe protein arrays with antibodies as well as the technical issues to consider when carrying out such experiments.

Applications of Protein Arrays for Small Molecule Drug Discovery and Characterization

The profiling of large chemical libraries produced using combinatorial chemistry techniques in high throughput assays has been considered to offer great promise in generating both more ’druggable’ targets x97 i.e. targets responsive to drugs x97 and lead compounds for treating disease. However, to date, this promise has not been realized. In many cases, high throughput screens were conducted with lipophilie small molecule libraries that failed to produce leads that finally made it to the clinic. This is perhaps unsurprising, since the task of developing a small molecule drug to combat a specific disease is a highly complicated, expensive, and time-consuming process, associated with a very high rate of failure. As a consequence, more attention is paid to in silico-based approaches during the early phase of drug discovery for both decreasing the costs and increasing the likelihood of producing so-called ’blockbuster’ drugs (van de Waterbeemd and Gifford, 2003). This review will summarize some of the challenges faced by the pharmaceutical industry, and will address how functional protein arrays can be used to address these challenges.

Corrigendum: SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.

Nat. Chem. Biol. 11, 511–517 (2015); published online 1 June 2015; corrected online 15 July 2015 and 11 February 2016 In the version of this article originally published online, the schematic for the construct in Figure 4a was incorrect. A corrected figure has been provided in the HTML and PDF versions of the article.