Biomedical subjects

Alexsandra Espejo

Publications and source records attributed to Alexsandra Espejo.

4 recordsLinked to original sources

PubMed2006

Tudor, MBT and chromo domains gauge the degree of lysine methylation.

The post-translational modification of histones regulates many cellular processes, including transcription, replication and DNA repair. A large number of combinations of post-translational modifications are possible. This cipher is referred to as the histone code. Many of the enzymes that lay down this code have been identified. However, so far, few code-reading proteins have been identified. Here, we describe a protein-array approach for identifying methyl-specific interacting proteins. We found that not only chromo domains but also tudor and MBT domains bind to methylated peptides from the amino-terminal tails of histones H3 and H4. Binding specificity observed on the protein-domain microarray was corroborated using peptide pull-downs, surface plasma resonance and far western blotting. Thus, our studies expose tudor and MBT domains as new classes of methyl-lysine-binding protein modules, and also demonstrates that protein-domain microarrays are powerful tools for the identification of new domain types that recognize histone modifications.

Histones↗

PubMed2004

Protein-domain microarrays.

Protein domains are independently folded regions of proteins that are often involved in protein-protein interactions. They are good candidates for the generation of protein microarrays because of their small size, their globular structures, and the fact that they are the protein-interacting workhorses of the cell. In addition, arrayed recombinant protein domains retain much of their binding specificity. Such microarrays can be probed with other recombinant proteins or fluorescently labeled peptides to identify potential binding partners and also determine how posttranslational modifications influence specific interactions. Thus, protein domains provide us with a system-oriented array that is focused on elucidating signal transduction pathway specificity and regulation.

Animals↗

PubMed2002

14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s).

Tuberin, the product of the tuberous sclerosis complex 2 tumor suppressor gene, is a phosphoprotein that negatively regulates phosphatidylinositol 3'-kinase signaling downstream of Akt. Several high stringency 14-3-3 binding sites that overlapped with Akt phosphorylation sites were identified in tuberin in silico. Recognition of tuberin by an alpha-14-3-3 binding site-specific antibody correlated with mitogen-induced phosphorylation of tuberin and recognition of tuberin by an alpha-Akt phosphorylation substrate antibody. Recognition of tuberin by both antibodies was blocked by inhibiting phosphatidylinositol 3'-kinase activity. Using a protein domain microarray, a tuberin peptide containing Ser(939) demonstrated phospho-specific binding to 14-3-3. Glutathione S-transferase pull-down assays with 14-3-3 fusion proteins revealed that all seven 14-3-3 isoforms (beta, gamma, zeta, epsilon, tau, eta, and sigma) could bind tuberin, and this interaction was abrogated by competition with phosphorylated but not unphosphorylated Ser(939) tuberin peptide. Tuberin also coimmunoprecipitated with 14-3-3, confirming the interaction between endogenous 14-3-3 and tuberin. These data establish the presence of functional and overlapping 14-3-3 and Akt recognition site(s) in tuberin.

14-3-3 Proteins↗

PubMed2002

A protein-domain microarray identifies novel protein-protein interactions.

Protein domains mediate protein-protein interactions through binding to short peptide motifs in their corresponding ligands. These peptide recognition modules are critical for the assembly of multiprotein complexes. We have arrayed glutathione S-transferase (GST) fusion proteins, with a focus on protein interaction domains, on to nitrocellulose-coated glass slides to generate a protein-domain chip. Arrayed protein-interacting modules included WW (a domain with two conserved tryptophans), SH3 (Src homology 3), SH2, 14.3.3, FHA (forkhead-associated), PDZ (a domain originally identified in PSD-95, DLG and ZO-1 proteins), PH (pleckstrin homology) and FF (a domain with two conserved phenylalanines) domains. Here we demonstrate, using peptides, that the arrayed domains retain their binding integrity. Furthermore, we show that the protein-domain chip can 'fish' proteins out of a total cell lysate; these domain-bound proteins can then be detected on the chip with a specific antibody, thus producing an interaction map for a cellular protein of interest. Using this approach we have confirmed the domain-binding profile of the signalling molecule Sam68 (Src-associated during mitosis 68), and have identified a new binding profile for the core small nuclear ribonucleoprotein SmB'. This protein-domain chip not only identifies potential binding partners for proteins, but also promises to recognize qualitative differences in protein ligands (caused by post-translational modification), thus getting at the heart of signal transduction pathways.

Amino Acid Sequence↗