Multi-monoubiquitylation controls VASP-mediated actin dynamics Icon for The Forest of Biologists

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics

Formation of a TBX20-CASZ1 protein complex is protective against dilated cardiomyopathy and critical for cardiac homeostasis

By the age of 40, one in five adults without symptoms of cardiovascular disease are at risk for developing congestive heart failure. Within this population, dilated cardiomyopathy (DCM) remains one of the leading causes of disease and death, with nearly half of cases genetically determined. Though genetic and high throughput sequencing-based approaches have identified sporadic and inherited mutations in a multitude of genes implicated in cardiomyopathy, how combinations of asymptomatic mutations lead to cardiac failure remains a mystery. Since a number of studies have implicated mutations of the transcription factor TBX20 in congenital heart diseases, we investigated the underlying mechanisms, using an unbiased systems-based screen to identify novel, cardiac-specific binding partners. We demonstrated that TBX20 physically and genetically interacts with the essential transcription factor CASZ1. This interaction is required for survival, as mice heterozygous for both Tbx20 and Casz1 die post-natally as a result of DCM. A Tbx20 mutation associated with human familial DCM sterically interferes with the TBX20-CASZ1 interaction and provides a physical basis for how this human mutation disrupts normal cardiac function. Finally, we employed quantitative proteomic analyses to define the molecular pathways mis-regulated upon disruption of this novel complex. Collectively, our proteomic, biochemical, genetic, and structural studies suggest that the physical interaction between TBX20 and CASZ1 is required for cardiac homeostasis, and further, that reduction or loss of this critical interaction leads to DCM. This work provides strong evidence that DCM can be inherited through a digenic mechanism

BIRC6 mediates imatinib resistance independently of Mcl-1

© 2017 Okumu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Baculoviral IAP repeat containing 6 (BIRC6) is a member of the inhibitors of apoptosis proteins (IAPs), a family of functionally and structurally related proteins that inhibit apoptosis. BIRC6 has been implicated in drug resistance in several different human cancers, however mechanisms regulating BIRC6 have not been extensively explored. Our phosphoproteomic analysis of an imatinib-resistant chronic myelogenous leukemia (CML) cell line (MYL-R) identified increased amounts of a BIRC6 peptide phosphorylated at S480, S482, and S486 compared to imatinib-sensitive CML cells (MYL). Thus we investigated the role of BIRC6 in mediating imatinib resistance and compared it to the well-characterized anti-apoptotic protein, Mcl-1. Both BIRC6 and Mcl-1 were elevated in MYL-R compared to MYL cells. Lentiviral shRNA knockdown of BIRC6 in MYL-R cells increased imatinib-stimulated caspase activation and resulted in a ~20-25-fold increase in imatinib sensitivity, without affecting Mcl-1. Treating MYL-R cells with CDK9 inhibitors decreased BIRC6 mRNA, but not BIRC6 protein levels. By contrast, while CDK9 inhibitors reduced Mcl-1 mRNA and protein, they did not affect imatinib sensitivity. Since the Src family kinase Lyn is highly expressed and active in MYL-R cells, we tested the effects of Lyn inhibition on BIRC6 and Mcl-1. RNAi-mediated knockdown or inhibition of Lyn (dasatinib/ponatinib) reduced BIRC6 protein stability and increased caspase activation. Inhibition of Lyn also increased formation of an N-terminal BIRC6 fragment in parallel with reduced amount of the BIRC6 phosphopeptide, suggesting that Lyn may regulate BIRC6 phosphorylation and stability. In summary, our data show that BIRC6 stability is dependent on Lyn, and that BIRC6 mediates imatinib sensitivity independently of Mcl-1 or CDK9. Hence, BIRC6 may be a novel target for the treatment of drugresistant CML where Mcl-1 or CDK9 inhibitors have failed

Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks

Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC-TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites illustrate the feasibility of this approach in quantitative phosphoproteomic studies

Addressing Obesity in Pregnancy: What Do Obstetric Providers Recommend?

Maternal obesity is associated with adverse pregnancy outcomes. To improve outcomes, obstetric providers must effectively evaluate and manage their obese pregnant patients. We sought to determine the knowledge, attitudes, and practice patterns of obstetric providers regarding obesity in pregnancy

THEMIS-SHP1 Recruitment by 4-1BB Tunes LCK-Mediated Priming of Chimeric Antigen Receptor-Redirected T Cells

Chimeric antigen receptor (CAR) T cell costimulation mediated by CD28 and 4-1BB is essential for CAR-T cell-induced tumor regression. However, CD28 and 4-1BB differentially modulate kinetics, metabolism and persistence of CAR-T cells, and the mechanisms governing these differences are not fully understood. We found that LCK recruited into the synapse of CD28-encoding CAR by co-receptors causes antigen-independent CAR-CD3z phosphorylation and increased antigen-dependent T cell activation. In contrast, the synapse formed by 4-1BB-encoding CAR recruits the THEMIS-SHP1 phosphatase complex that attenuates CAR-CD3z phosphorylation. We further demonstrated that the CAR synapse can be engineered to recruit either LCK to enhance the kinetics of tumor killing of 4-1BB CAR-T cells or SHP1 to tune down cytokine release of CD28 CAR-T cells

Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy

Immunotherapy holds tremendous promise for improving cancer treatment1. Administering radiotherapy with immunotherapy has been shown to improve immune responses and can elicit an “abscopal effect”2. Unfortunately, response rates for this strategy remain low3. Herein, we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NPs formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent upon NP surface properties. We showed that AC-NPs deliver tumor specific proteins to antigen-presenting cells and significantly improve the efficacy of αPD-1 treatment using the B16F10 melanoma model, generating up to 20% cure rate as compared to 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+/Treg and CD8+/Treg ratios. Our work presents a novel strategy for improving cancer immunotherapy with nanotechnology

Kinome Profiling Identifies Druggable Targets for Novel Human Cytomegalovirus (HCMV) Antivirals

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC 50 values of 3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs

Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation