The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions

The focal adhesion protein testin is a modular scaffold and tumour suppressor that consists of an N-terminal cysteine rich (CR) domain, a PET domain of unknown function and three C-terminal LIM domains. Testin has been proposed to have an open and a closed conformation based on the observation that its N-terminal half and C-terminal half directly interact. Here we extend the testin conformational model by demonstrating that testin can also form an antiparallel homodimer. In support of this extended model we determined that the testin region (amino acids 52-233) harbouring the PET domain interacts with the C-terminal LIM1-2 domains in vitro and in cells, and assign a critical role to tyrosine 288 in this interaction

Eliminating oncogenic RAS: back to the future at the drawing board.

RAS drug development has made enormous strides in the past ten years, with the first direct KRAS inhibitor being approved in 2021. However, despite the clinical success of covalent KRAS-G12C inhibitors, we are immediately confronted with resistances as commonly found with targeted drugs. Previously believed to be undruggable due to its lack of obvious druggable pockets, a couple of new approaches to hit this much feared oncogene have now been carved out. We here concisely review these approaches to directly target four druggable sites of RAS from various angles. Our analysis focuses on the lessons learnt during the development of allele-specific covalent and non-covalent RAS inhibitors, the potential of macromolecular binders to facilitate the discovery and validation of targetable sites on RAS and finally an outlook on a future that may engage more small molecule binders to become drugs. We foresee that the latter could happen mainly in two ways: First, non-covalent small molecule inhibitors may be derived from the development of covalent binders. Second, reversible small molecule binders could be utilized for novel targeting modalities, such as degraders of RAS. Provided that degraders eliminate RAS by recruiting differentially expressed E3-ligases, this approach could enable unprecedented tissue- or developmental stage-specific destruction of RAS with potential advantages for on-target toxicity. We conclude that novel creative ideas continue to be important to exterminate RAS in cancer and other RAS pathway-driven diseases, such as RASopathies

Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion

Bruceine D Identified as a Drug Candidate against Breast Cancer by a Novel Drug Selection Pipeline and Cell Viability Assay.

The multi-target effects of natural products allow us to fight complex diseases like cancer on multiple fronts. Unlike docking techniques, network-based approaches such as genome-scale metabolic modelling can capture multi-target effects. However, the incompleteness of natural product target information reduces the prediction accuracy of in silico gene knockout strategies. Here, we present a drug selection workflow based on context-specific genome-scale metabolic models, built from the expression data of cancer cells treated with natural products, to predict cell viability. The workflow comprises four steps: first, in silico single-drug and drug combination predictions; second, the assessment of the effects of natural products on cancer metabolism via the computation of a dissimilarity score between the treated and control models; third, the identification of natural products with similar effects to the approved drugs; and fourth, the identification of drugs with the predicted effects in pathways of interest, such as the androgen and estrogen pathway. Out of the initial 101 natural products, nine candidates were tested in a 2D cell viability assay. Bruceine D, emodin, and scutellarein showed a dose-dependent inhibition of MCF-7 and Hs 578T cell proliferation with IC(50) values between 0.7 to 65 μM, depending on the drug and cell line. Bruceine D, extracted from Brucea javanica seeds, showed the highest potency

Beta3 integrins: major therapeutic targets of the near future.

Integrins are major cell adhesion receptors which assume two important functions: first they act as anchoring molecules allowing firm cellular attachment to the extracellular matrix, and second they work as signalling receptors being able to transduce signals in both directions (outside-in and inside-out) through the plasma membrane. Their biological importance is determined by their involvement in many physiological phenomena. Furthermore, their implication in various diseases and their accessibility to drugs make them interesting therapeutic targets

RGD, the Rho'd to cell spreading

Some RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding. However, the precise involvement of each of these recognition sites during cell adhesion is still unclear. Here we review recent investigations on integrin alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen providing evidence that the fibrinogen synergy gamma(400-411) sequence by itself promotes cell attachment by initiating alphaIIbbeta3 clustering and recruitment of intracellular proteins to focal complexes, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to induce a conformational change necessary for RhoA activation and full cell spreading

Evidence from site-directed mutagenesis that the cytoplasmic domain of the beta3 subunit influences the conformational state of the alphaVbeta3 integrin ectodomain.

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor alphavbeta3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on beta3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire beta3 cytoplasmic domain induced constitutive LIBS exposure on alphavbeta3 and alphaIIbeta3. Deletion of the C-terminal NITY759 sequence or disruption of the NPLY747 motif by a Y747A substitution impaired extracellular conformational changes on alphavbeta3 following RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn2+-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY747 motif in the beta3 cytoplasmic domain, rather than potential phosphorylation of Tyr747 or Tyr759, is a prerequisite for conformational changes within the alphavbeta3 ectodomain, and suggest that two different mechanisms are responsible for RGDS- and Mn2+-dependent conformational changes

A new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin alphaIIbbeta3-dependent RhoA activation during cell spreading

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2

Distinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events