Hsp83 loss suppresses proteasomal activity resulting in an upregulation of caspase-dependent compensatory autophagy

The 2 main degradative pathways that contribute to proteostasis are the ubiquitin-proteasome system and autophagy but how they are molecularly coordinated is not well understood. Here, we demonstrate an essential role for an effector caspase in the activation of compensatory autophagy when proteasomal activity is compromised. Functional loss of Hsp83, the Drosophila ortholog of human HSP90 (heat shock protein 90), resulted in reduced proteasomal activity and elevated levels of the effector caspase Dcp-1. Surprisingly, genetic analyses showed that the caspase was not required for cell death in this context, but instead was essential for the ensuing compensatory autophagy, female fertility, and organism viability. The zymogen pro-Dcp-1 was found to interact with Hsp83 and undergo proteasomal regulation in an Hsp83-dependent manner. Our work not only reveals unappreciated roles for Hsp83 in proteasomal activity and regulation of Dcp-1, but identifies an effector caspase as a key regulatory factor for sustaining adaptation to cell stress in vivo

Streamlining T cell engager development with a diverse panel of fully human CD3-binding antibodies, bispecific engineering technology, and an integrated discovery engine

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Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi

The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2)mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087), was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-α3 and Smr) with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2)mbn cells (p<0.05) following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival

Caspase regulation of autophagy in Drosophila melanogaster

Autophagy is an evolutionary conserved process whereby intracellular components are sequestered and delivered to lysosomes for degradation. Autophagy acts as a cell survival mechanism in response to stress, such as starvation, and also engages in a complex relationship with apoptosis. Understanding the crosstalk between autophagy and apoptosis is important, as it plays a critical role in the balance between survival and death, and has important implications in both normal development and human diseases. To better understand the crosstalk between autophagy and apoptosis, I examined the role of the Drosophila melanogaster effector caspase Dcp-1 in starvation-induced autophagy during mid-oogenesis. I confirmed that Dcp-1 positively regulates starvation-induced autophagic flux in degenerating mid-stage egg chambers, and does so in a catalytically dependent manner. Dcp-1 candidate interactors/substrates, identified previously, were analyzed using in vitro autophagy assays to elucidate potential mechanisms related to Dcp-1-mediated autophagy. I identified 13 novel Dcp-1-associated regulators of starvation-induced autophagy, including the chloride intracellular channel protein Clic, the heat shock protein Hsp83, and the mitochondrial protein SesB. In vivo analyses revealed that Clic and Hsp83 act as negative regulators of autophagic flux following starvation during Drosophila oogenesis. Further investigation into the possible mitochondrial-related role of Dcp-1 in autophagy revealed that Dcp-1 partially localizes within the mitochondria where it functions to regulate mitochondrial network morphology and ATP levels, demonstrated both in vitro and in vivo during mid-oogenesis. Moreover, I found that the pro-form of Dcp-1 interacts with the adenine nucleotide translocase SesB, and as such, Dcp-1 does not cleave SesB but rather affects its stability. In addition, I identified SesB as a novel negative regulator of autophagic flux during mid-oogenesis. Depletion of ATP or reduction of SesB levels rescued the autophagic defect in Dcp-1 loss-of-function flies, and genetic interaction studies revealed that SesB acts downstream of Dcp-1 in the regulation of autophagy. In conclusion, I found that non-apoptotic caspase activity is an important molecular mechanism underlying autophagy regulation and mitochondrial physiology in vivo, and have provided a foundation for further analyses involving Dcp-1-associated regulators of starvation-induced autophagy

Pan-Specific and Partially Selective Dye-Labeled Peptidic Inhibitors of the Polycomb Paralog Proteins

Epigenetic regulation of gene expression is in part controlled by post-translational modifications on histone proteins. Histone methylation is a key epigenetic mark that controls gene transcription and repression. There are five human polycomb paralog proteins (Cbx2/4/6/7/8) which use their chromodomains to recognize trimethylated lysine 27 on Histone 3 (H3K27me3). Recognition of the methyllysine side chain is achieved through multiple cation-pi interactions within an ‘aromatic cage’ motif. Despite high structural similarity within the chromodomains of this protein family, they each have unique functional roles and are linked to different cancers. Selective inhibition of different CBX proteins is highly desirable for both fundamental studies and potential therapeutic applications. We will report on a series of peptidic inhibitors that selectively target certain polycomb paralogs. We have identified peptidic scaffolds with sub-micromolar potency, and will report examples that are pan-specific and that are partially selective for individual members within the family. The data presented include extensive new synthesis with characterization by LC, Mass Spectrometry, and NMR. The binding interactions are measured by fluorescence polarization, molecular dynamics simulations, and protein microarray assays. These results highlight important structure-activity relationships that allow for selectivity to be achieved through interactions outside of the methyllysine binding aromatic cage motif.</div

<i>Ex Vivo</i> Detection of Circulating Tumor Cells from Whole Blood by Direct Nanoparticle Visualization

The detection of circulating tumor cells (CTCs) from blood samples can predict prognosis, response to systemic chemotherapy, and metastatic spread of carcinoma. Therefore, approaches for CTC identification is an important aspect of current cancer research. Here, a method for the direct visualization of nanoparticle-coated CTCs under dark field illumination is presented. A metastatic breast cancer cell line (4T1) was transduced with a non-native target protein (Thy1.1). Positive 4T1-Thy1.1 cells incubated with antibody-coated metallic nanoshells appeared overly bright at low magnification, allowing a quick screening of samples and easy visual detection of even single isolated CTCs. The use of a nontransduced cell line as control creates the ideal scenario to evaluate nonspecific binding. A murine metastatic tumor model with the 4T1-Thy1.1 cell line was also implemented. Blood was drawn from mice over the course of one month, and CTCs were successfully detected in all positive subjects. This work validates the use of metallic nanoshells as labels for direct visualization of CTCs while providing guidelines to a systematic development of nanotechnology-based detection systems for CTCs

The Drosophila TIPE family member Sigmar interacts with the Ste20-like kinase Misshapen and modulates JNK signaling, cytoskeletal remodeling and autophagy

TNFAIP8 and other mammalian TIPE family proteins have attracted increased interest due to their associations with disease-related processes including oncogenic transformation, metastasis, and inflammation. The molecular and cellular functions of TIPE family proteins are still not well understood. Here we report the molecular and genetic characterization of the Drosophila TNFAIP8 homolog, CG4091/sigmar. Previous gene expression studies revealed dynamic expression of sigmar in larval salivary glands prior to histolysis. Here we demonstrate that in sigmar loss-of-function mutants, the salivary glands are morphologically abnormal with defects in the tubulin network and decreased autophagic flux. Sigmar localizes subcellularly to microtubule-containing projections in Drosophila S2 cells, and co-immunoprecipitates with the Ste20-like kinase Misshapen, a regulator of the JNK pathway. Further, the Drosophila TNF ligand Eiger can induce sigmar expression, and sigmar loss-of-function leads to altered localization of pDJNK in salivary glands. Together, these findings link Sigmar to the JNK pathway, cytoskeletal remodeling and autophagy activity during salivary gland development, and provide new insights into TIPE family member function