Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders.

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver.

Structural basis for Rab6 activation by the Ric1-Rgp1 complex.

Rab GTPases act as molecular switches to regulate organelle homeostasis and membrane trafficking. Rab6 plays a central role in regulating cargo flux through the Golgi and is activated via nucleotide exchange by the Ric1-Rgp1 protein complex. Ric1-Rgp1 is conserved throughout eukaryotes but the structural and mechanistic basis for its function has not been established.

CryoDRGN-ET: deep reconstructing generative networks for visualizing dynamic biomolecules inside cells.

Advances in cryo-electron tomography (cryo-ET) have produced new opportunities to visualize the structures of dynamic macromolecules in native cellular environments. While cryo-ET can reveal structures at molecular resolution, image processing algorithms remain a bottleneck in resolving the heterogeneity of biomolecular structures in situ. Here, we introduce cryoDRGN-ET for heterogeneous reconstruction of cryo-ET subtomograms.

Structural basis for Rab6 activation by the Ric1-Rgp1 complex.

Rab GTPases act as molecular switches to regulate organelle homeostasis and membrane trafficking. Rab6 plays a central role in regulating cargo flux through the Golgi and is activated via nucleotide exchange by the Ric1-Rgp1 protein complex. Ric1-Rgp1 is conserved throughout eukaryotes but the structural and mechanistic basis for its function has not been established.

A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma.

Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic xenografts derived from patients with GBM. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators.

Epigenomics may begin to explain differential response to hypomethylating agents in MMR-D hypermethylated endometrial cancer.

This work examines differences in chromatin accessibility, methylation, and response to DNA hypomethylating agents between mismatch repair-deficient and non-mismatch repair-deficient endometrial cancer. Next-generation sequencing of a stage 1B, grade 2 endometrioid endometrial cancer tumor revealed microsatellite instability and a variant of unknown significance in along with global and hypermethylation. Inhibition of viability by decitabine in the study and comparison tumors was minimal, as shown by an inhibitory effect of 0 and 17.

Structure of the RhlR-PqsE complex from Pseudomonas aeruginosa reveals mechanistic insights into quorum-sensing gene regulation.

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P. aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers.

The structural basis of Salmonella AB toxin neutralization by antibodies targeting the glycan-receptor binding subunits.

Many bacterial pathogens secrete AB toxins comprising two functionally distinct yet complementary "A" and "B" subunits to benefit the pathogens during infection. The lectin-like pentameric B subunits recognize specific sets of host glycans to deliver the toxin into target host cells. Here, we offer the molecular mechanism by which neutralizing antibodies, which have the potential to bind to all glycan-receptor binding sites and thus completely inhibit toxin binding to host cells, are inhibited from exerting this action.

Experimental evaluation of super-resolution imaging and magnification choice in single-particle cryo-EM.

The resolution of cryo-EM reconstructions is fundamentally limited by the Nyquist frequency, which is half the sampling frequency of the detector and depends upon the magnification used. In principle, super-resolution imaging should enable reconstructions to surpass the physical Nyquist limit by increasing sampling frequency, yet there are few reports of reconstructions that do so. Here we directly examine the contribution of super-resolution information, obtained with the K3 direct electron detector using a 2-condenser microscope, to single-particle cryo-EM reconstructions surpassing the physical Nyquist limit.

PLEKHA7, an Apical Adherens Junction Protein, Suppresses Inflammatory Breast Cancer in the Context of High E-Cadherin and p120-Catenin Expression.

Inflammatory breast cancer is a highly aggressive form of breast cancer that forms clusters of tumor emboli in dermal lymphatics and readily metastasizes. These cancers express high levels of E-cadherin, the major mediator of adherens junctions, which enhances formation of tumor emboli. Previous studies suggest that E-cadherin promotes cancer when the balance between apical and basolateral cadherin complexes is disrupted.

Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly.

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD).

Integration of Comprehensive Genomic Analysis and Functional Screening of Affected Molecular Pathways to Inform Cancer Therapy.

Objective: To select optimal therapies based on the detection of actionable genomic alterations in tumor samples is a major challenge in precision medicine.

Methods: We describe an effective process (opened December 1, 2017) that combines comprehensive genomic and transcriptomic tumor profiling, custom algorithms and visualization software for data integration, and preclinical 3-dimensiona ex vivo models for drug screening to assess response to therapeutic agents targeting specific genomic alterations. The process was applied to a patient with widely metastatic, weakly hormone receptor positive, HER2 nonamplified, infiltrating lobular breast cancer refractory to standard therapy.

A phase 1 and randomized, placebo-controlled phase 2 trial of bevacizumab plus dasatinib in patients with recurrent glioblastoma: Alliance/North Central Cancer Treatment Group N0872.

Background: Src signaling is markedly upregulated in patients with invasive glioblastoma (GBM) after the administration of bevacizumab. The Src family kinase inhibitor dasatinib has been found to effectively block bevacizumab-induced glioma invasion in preclinical models, which led to the hypothesis that combining bevacizumab with dasatinib could increase bevacizumab efficacy in patients with recurrent GBM.

Methods: After the completion of the phase 1 component, the phase 2 trial (ClinicalTrials.

Classification of the human phox homology (PX) domains based on their phosphoinositide binding specificities.

Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry.

Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis.

Lipid droplets (LDs) serve as cytoplasmic reservoirs for energy-rich fatty acids (FAs) stored in the form of triacylglycerides (TAGs). During nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is coordinated remains poorly understood. The ER protein Mdm1 is a molecular tether that plays a role in clustering LDs during nutrient depletion, but its mechanism of function remains unknown.

Efficacy of the MDM2 Inhibitor SAR405838 in Glioblastoma Is Limited by Poor Distribution Across the Blood-Brain Barrier.

Controversy exists surrounding whether heterogeneous disruption of the blood-brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM.

Lipid droplet biogenesis is spatially coordinated at ER-vacuole contacts under nutritional stress.

Eukaryotic cells store lipids in cytosolic organelles known as lipid droplets (LDs). Lipid droplet bud from the endoplasmic reticulum (ER), and may be harvested by the vacuole for energy during prolonged periods of starvation. How cells spatially coordinate LD production is poorly understood.

Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling.

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7.

Male Infertility Is Responsible for Nearly Half of the Extinction Observed in the Mouse Collaborative Cross.

The goal of the Collaborative Cross (CC) project was to generate and distribute over 1000 independent mouse recombinant inbred strains derived from eight inbred founders. With inbreeding nearly complete, we estimated the extinction rate among CC lines at a remarkable 95%, which is substantially higher than in the derivation of other mouse recombinant inbred populations. Here, we report genome-wide allele frequencies in 347 extinct CC lines.

Assessment of Tumor Heterogeneity, as Evidenced by Gene Expression Profiles, Pathway Activation, and Gene Copy Number, in Patients with Multifocal Invasive Lobular Breast Tumors.

Background: Invasive lobular carcinoma (ILC) comprises approximately ~10-20% of breast cancers. In general, multifocal/multicentric (MF/MC) breast cancer has been associated with an increased rate of regional lymph node metastases. Tumor heterogeneity between foci represents a largely unstudied source of genomic variation in those rare patients with MF/MC ILC.

Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity.

E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell-cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent.

Src family kinases differentially influence glioma growth and motility.

Src-family kinase (SFK) signaling impacts multiple tumor-related properties, particularly in the context of the brain tumor glioblastoma. Consequently, the pan-SFK inhibitor dasatinib has emerged as a therapeutic strategy, despite physiologic limitations to its effectiveness in the brain. We investigated the importance of individual SFKs (Src, Fyn, Yes, and Lyn) to glioma tumor biology by knocking down individual SFK expression both in culture (LN229, SF767, GBM8) and orthotopic xenograft (GBM8) contexts.

The Rho guanine nucleotide exchange factor Syx regulates the balance of dia and ROCK activities to promote polarized-cancer-cell migration.

The role of RhoA in promoting directed cell migration has been complicated by studies showing that it is activated both in the front and the rear of migrating cells. We report here that the RhoA-specific guanine nucleotide exchange factor Syx is required for the polarity of actively migrating brain and breast tumor cells. This function of Syx is mediated by the selective activation of the RhoA downstream effector Dia1, the subsequent reorganization of microtubules, and the downregulation of focal adhesions and actin stress fibers.

TEM4 is a junctional Rho GEF required for cell-cell adhesion, monolayer integrity and barrier function.

Signaling events mediated by Rho family GTPases orchestrate cytoskeletal dynamics and cell junction formation. The activation of Rho GTPases is tightly regulated by guanine-nucleotide-exchange factors (GEFs). In this study, we identified a novel Rho-specific GEF called TEM4 (tumor endothelial marker 4) that associates with multiple members of the cadherin-catenin complex and with several cytoskeleton-associated proteins.

Paneth cell antimicrobial peptides: topographical distribution and quantification in human gastrointestinal tissues.

Antimicrobial peptides and proteins are key effectors of innate immunity, expressed both by circulating phagocytic cells and by epithelial cells of mucosal tissues. In the human small intestine, Paneth cells are secretory epithelial cells that express the antimicrobials human alpha-defensin-5 (HD5), HD6, lysozyme and secretory phospholipase A(2) (sPLA(2)), and recent studies have implicated reduced HD5 and HD6 expression levels in the pathogenesis of ileal Crohn's disease. However, expression levels of these molecules have not been determined routinely by techniques that readily permit quantitative comparisons of their distribution between tissues and samples.