Faculty Bibliography

The unfolded protein response (UPR) is an intracellular signaling pathway that is activated in response to stress in the endoplasmic reticulum (ER). UPR can effectively cope with stress by reducing the amount of misfolded protein overload in this subcellular organelle. Significantly, ER-stress is associated with various neurodegenerative disorders, diabetes and cancer, where UPR affects the course of disease manifestation in many cases. While significant progress has been made in various experimental systems over the years, suitable models for in vivo analyses of UPR and disease remain scarce. In this regard, recent developments of Drosophila markers and genetic tools for UPR studies provide powerful means to investigate the connection between UPR and disease in vivo. Here, we review the molecular components of the Drosophila UPR as well as the disease models that may be affected by this signaling pathway

Stress in the endoplasmic reticulum (ER stress) and its cellular response, the unfolded protein response (UPR), are implicated in a wide variety of diseases, but its significance in many disorders remains to be validated in vivo. Here, we analyzed a branch of the UPR mediated by xbp1 in Drosophila to establish its role in neurodegenerative diseases. The Drosophila xbp1 mRNA undergoes ire-1-mediated unconventional splicing in response to ER stress, and this property was used to develop a specific UPR marker, xbp1-EGFP, in which EGFP is expressed in frame only after ER stress. xbp1-EGFP responds specifically to ER stress, but not to proteins that form cytoplasmic aggregates. The ire-1/xbp1 pathway regulates heat shock cognate protein 3 (hsc3), an ER chaperone. xbp1 splicing and hsc3 induction occur in the retina of ninaE(G69D)-/+, a Drosophila model for autosomal dominant retinitis pigmentosa (ADRP), and reduction of xbp1 gene dosage accelerates retinal degeneration of these animals. These results demonstrate the role of the UPR in the Drosophila ADRP model and open new opportunities for examining the UPR in other Drosophila disease models

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Here, we show that apoptotic cells express the secretory factors wingless (wg) and decapentaplegic (dpp). When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation was observed. Significantly, wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, we provide evidence that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation

In Drosophila, differences between segments, such as the presence or absence of appendages, are controlled by Hox transcription factors. The Hox protein Ultrabithorax (Ubx) suppresses limb formation in the abdomen by repressing the leg selector gene Distalless, whereas Antennapedia (Antp), a thoracic Hox protein, does not repress Distalless. We show that the Hox cofactors Extradenticle and Homothorax selectively enhance Ubx, but not Antp, binding to a Distalless regulatory sequence. A C-terminal peptide in Ubx stimulates binding to this site. However, DNA binding is not sufficient for Distalless repression. Instead, an additional alternatively spliced domain in Ubx is required for Distalless repression but not DNA binding. Thus, the functional specificities of Hox proteins depend on both DNA binding-dependent and -independent mechanisms

Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Here, we report that Drosophila melanogaster IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. We show that Rpr, but not Hid (head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, we found ubcD1, which suppresses rpr-induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro. Our results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting its degradation

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA

We characterize a 37-bp element (fkh[250]) derived from the fork head (fkh) gene, a natural target of the Hox gene Sex combs reduced (Scr). In vitro, Scr cooperatively binds to this DNA with the Hox cofactor Extradenticle (Exd), and the activation of this enhancer in vivo requires Scr and exd. Other Hox/Exd heterodimers do not activate this element in vivo and do not bind this element with high affinity in vitro. The amino-terminal arm of the Scr homeodomain is crucial for the specific activation of this element in vivo. By mutating two base pairs within this element, we can convert the Scr/Exd-binding site to a Hox/Exd consensus site that binds several different Hox/Exd heterodimers. This element, fkh[250(con)], is activated by Scr, Antennapedia (Antp), and Ultrabithorax (Ubx) but repressed by abdominal-A (abd-A). We also show that Scr and Exd are only able to activate the fkh[250] element during the early stages of embryogenesis because, by stage 11, Scr negatively regulates the gene homothorax (hth), which is required for the nuclear localization of Exd. These results suggest that Exd is a specificity cofactor for the trunk Hox genes, and that the control of Exd subcellular localization is a mechanism to regulate Hox activity during development