Peptide Ligands for Pro-survival Protein Bfl-1 from Computationally Guided Library Screening

Pro-survival members of the Bcl-2 protein family inhibit cell death by binding short helical BH3 motifs in pro-apoptotic proteins. Mammalian pro-survival proteins Bcl-x[subscript L], Bcl-2, Bcl-w, Mcl-1, and Bfl-1 bind with varying affinities and specificities to native BH3 motifs, engineered peptides, and small molecules. Biophysical studies have determined interaction patterns for these proteins, particularly for the most-studied family members Bcl-x[subscript L] and Mcl-1. Bfl-1 is a pro-survival protein implicated in preventing apoptosis in leukemia, lymphoma, and melanoma. Although Bfl-1 is a promising therapeutic target, relatively little is known about its binding preferences. We explored the binding of Bfl-1 to BH3-like peptides by screening a peptide library that was designed to sample a high degree of relevant sequence diversity. Screening using yeast-surface display led to several novel high-affinity Bfl-1 binders and to thousands of putative binders identified through deep sequencing. Further screening for specificity led to identification of a peptide that bound to Bfl-1 with K[subscript d] < 1 nM and very slow dissociation from Bfl-1 compared to other pro-survival Bcl-2 family members. A point mutation in this sequence gave a peptide with ~50 nM affinity for Bfl-1 that was selective for Bfl-1 in equilibrium binding assays. Analysis of engineered Bfl-1 binders deepens our understanding of how the binding profiles of pro-survival proteins differ and may guide the development of targeted Bfl-1 inhibitors.National Institute of General Medical Sciences (U.S.) (Award GM084181)National Institute of General Medical Sciences (U.S.) (Award P50-GM68762

Protein 3D Structure Computed from Evolutionary Sequence Variation

The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing

Sequence Motifs in MADS Transcription Factors Responsible for Specificity and Diversification of Protein-Protein Interaction

Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution

Abnormalities of in vitro immunoglobulin synthesis by peripheral blood lymphocytes from patients with essential mixed cryoglobulinemia

Peripheral blood mononuclear cells from patients with essential mixed cryoglobulinemia (EMC) were studied for their ability to differentiate into cells containing cytoplasmic immunoglobulins (Ig) and to synthetize Ig after in vitro pokeweed mitogen activation. EMC lymphocytes showed a significant defective differentiation and Ig synthesis compared to normal controls. Coculture experiments carried out mixing enriched normal T- and EMC B-cell suspensions, and vice versa, showed that (a) the EMC B-cell-defective Ig synthesis still persisted after removal of suppressor activity by irradiation, both with autologous and with normal allogeneic T suspensions and (b) EMC T cells displayed a less efficient activity in helping Ig production by normal B lymphocytes. A comparable, reduced response was also found after activation with Staphylococcus aureus strain Cowan I. Taken together these results seem to indicate that in essential mixed cryoglobulinemia an impaired T-cell helper activity coexists with a B-lymphocyte impairment. The significance of these abnormalities in the pathogenesis of EMC is discussed

Red-emitting chimeric firefly luciferase for in\uc2\ua0vivo imaging in low ATP cellular environments

Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Kmvalues for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Kmvalue for ATP similar to CBR and \ue2\u88\ubc2.6-fold higher specific activity. The red-emitting PLR3 was \ue2\u88\ubc2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Kmvalue in low ATP environments