Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma.

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind.

A High-Throughput Screen for Transcription Activation Domains Reveals Their Sequence Features and Permits Prediction by Deep Learning.

Acidic transcription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that promote their dynamic behavior, "fuzzy" interactions, and target specificity. We screened a large set of random 30-mer peptides for AD function in yeast and trained a deep neural network (ADpred) on the AD-positive and -negative sequences. ADpred identifies known acidic ADs within transcription factors and accurately predicts the consequences of mutations.

Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.

Deletions within genes coding for subunits of the transcription coactivator SAGA caused strong genome-wide defects in transcription and SAGA-mediated chromatin modifications. In contrast, rapid SAGA depletion produced only modest transcription defects at 13% of protein-coding genes - genes that are generally more sensitive to rapid TFIID depletion. However, transcription of these 'coactivator-redundant' genes is strongly affected by rapid depletion of both factors, showing the overlapping functions of TFIID and SAGA at this gene set.

RAS/Effector Interactions from Structural and Biophysical Perspective.

RAS is a molecular switch that regulates a large number of pathways through interactions with many effector proteins. Most RAS/effector complexes are short-lived, demonstrating fast association and fast dissociation rate and Kds ranging from 10(-8)-10(-5) M, compatible with the signaling function of these interactions in the cell. RAS effectors share little sequence homology but all contain an RAS binding domain that exhibits ubiquitin fold.

How structure defines affinity in protein-protein interactions.

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success.

Alteration of the C-terminal ligand specificity of the erbin PDZ domain by allosteric mutational effects.

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain.

A single-tube assembly of DNA using the transfer-PCR (TPCR) platform.

DNA cloning is a basic methodology employed for multiple applications in all life-science disciplines. In order to facilitate DNA cloning we developed Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit.

Computational methods for controlling binding specificity.

Learning to control, protein-binding specificity is useful for both fundamental and applied biology. In fundamental research, better understanding of complicated signaling networks could be achieved through engineering of regulator proteins to bind to only a subset of their effector proteins. In applied research such as drug design, nonspecific binding remains a major reason for failure of many drug candidates.

Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering.

DNA cloning and protein engineering are basic methodologies employed for various applications in all life-science disciplines. Manipulations of DNA however, could be a lengthy process that slows down subsequent experiments. To facilitate both DNA cloning and protein engineering, we present Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector.

Multispecific recognition: mechanism, evolution, and design.

Accumulating evidence shows that many particular proteins have evolved to bind multiple targets, including other proteins, peptides, DNA, and small molecule substrates. Multispecific recognition might be not only common but also necessary for the robustness of signaling and metabolic networks in the cell. It is also important for the immune response and for regulation of transcription and translation.