Hermann Steller - US grants

Apoptosis is a morphologically distinct from of programmed cell death that plays important roles in development, tissue homoestasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders. It is now clear that apoptosis occurs by activating an intrinsic cell suicide program which is constitutively expressed in most animal cells, and that key components of this program have been conserved in evolution from worms to insects to man. A central step in the execution of apoptosis is the activation of an unusual class of cysteine proteases, termed caspases, that are widely expressed as inactive zymogens. The overall objective of the proposed research is to gain insight into the molecular mechanisms that control caspase activation and cell death. The specific goals of this proposal are to characterize wild type and mutant versions of genes that were originally identified in genetic screens for cell death modifiers in Drosophila. We will use a multidisciplinary approach that integrates genetics, biochemistry, cell biology, Drosophila gene transfer and mammalian cell transfection experiments to elucidate the mechanism by which the pro-apoptotic proteins REAPER, HID (head involution defective) and GRIM kill. In particular, we will study functional and biochemical interactions between REAPER, HID and GRIM with Drosophila and human inhibitor of apoptosis proteins (IAPs). We also propose to characterize several novel genes corresponding to mutants that strongly affect REAPER, HID and GRIM-induced cell death, and to define their precise role and mechanism of action during apoptosis. Finally, we will investigate to what extent the apoptotic pathway has been conserved between Drosophila and mammals. This work should significantly advance our understanding of how apoptosis is regulated, and how this process can be manipulated for therapeutic purposes.

Apoptosis is a morphologically distinct from of programmed cell death that plays important roles in development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders. Mitochondria have a key function for; sensing and propagating cell death signals. For example, the release of cytochrome c from mitochondria into the cytoplasm is thought to be a critical step for activating the cell death program. A new mitochondrial protein, termed-ARTS (Apoptosis Related protein in the TGF-beta Signaling pathway), appears to mediate the induction of apoptosis in mammalian cells in response to several distinct death-inducing stimuli. ARTS is a novel septin protein family member that translocates; from mitochondria to the nucleus at the onset of apoptosis. Furthermore, a Drosophila homolog of ARTS, encoded by the peanut locus, is required for the induction of cell death by the pro-apoptotic genes reaper, hid and grim in this organism. The overall objective of the proposed research is to use an integrated approach that combines Drosophila genetics and molecular biology with mammalian cell culture experiments to gain insight into the mechanism by which ARTS induces apoptosis. We will address the following specific questions: 1. How conserved is the pro-apoptotic function of human ARTS and Drosophila PEANUT? 2. What is the role of ARTS for cell killing by the pro-apoptotic proteins Reaper, Hid and Grim in mammalian cells? 3. Where in the cell does ARTS act to induce apoptosis? 4. Are there functional and biochemical interactions between ARTS/peanut with IAPS (inhibitor of apoptosis proteins) and Sina/Siah proteins? This work should significantly advance our understanding of how apoptosis is regulated, and how this process can be manipulated for therapeutic purposes.

Apoptosis is a morphologically distinct form of programmed cell death that plays important roles in development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders. It is now clear that apoptosis occurs by activating an intrinsic cell suicide program which is constitutively expressed in most animal cells, and that key components of this program have been conserved in evolution from worms to insects to man. A central step in the execution of apoptosis is the activation of a specific class of cysteine proteases, termed caspases, that are widely expressed as inactive zymogens. The overall objective of the proposed research is to gain insight into the molecular mechanisms that control caspase activation and cell death. Work during the previous project period has demonstrated that a set of ubiquitin pathway proteins play a complex but specific role in regulating the onset of apoptosis via selective protein degradation. In particular, Inhibitor of Apoptosis Proteins (IAPs) can ubiquitinate certain caspases in live cells, but auto-ubiquitinate and self-destruct in cells that are doomed to die. The specific goals of this proposal are to define the mechanism by which Reaper-family protein stimulate the auto-ubiquitination of IAPs in both Drosophila and mammalian cells, and the role of this pathway for the regulation of cell death in both normal and cancer cells. Furthermore, we will test the hypothesis that defects in IAP self-conjugation contribute to cell immortalization and malignancy. For this project, we will use a multidisciplinary approach that integrates Drosophila genetics, biochemistry, cell biology, and mammalian cell culture studies. As part of this study, we propose to generate small, cell permeable peptide derivatives of Reaper ("Reaper-mimetics') to inactivate cellular IAPs ("protein knock-outs") and examine their role in the regulation of mammalian cell death. This work should significantly advance our understanding of how apoptosis is regulated, and how this process can be manipulated for therapeutic purposes.

DESCRIPTION (provided by applicant): The selective degradation of intracellular proteins is of central importance for the generation, function and survival of eukaryotic cells. The ubiquitin-proteasome system (UPS) is responsible for the controlled degradation of most intracellular proteins, and abnormal regulation of the UPS is associated with a variety of human diseases, including cancer, myopathies, and neurodegenerative disorders. Although dramatic progress has been made in understanding the structure and function of proteasomes, we still know extremely little about how proteasome activity is dynamically regulated in time and space. The activity of the 26S proteasome declines with age, but the underlying molecular mechanisms remain unknown. Our prior work focused on the regulation of caspases by the UPS, and results obtained during the current funding cycle revealed the joint use of proteasomes and caspases in the controlled demolition of cellular structures that is needed for terminal sperm differentiatio in Drosophila. Similar mechanisms are thought to mediate the remodeling of other cell types, including neurons and muscle, in both insects and vertebrates. The overall goal of this project is to understand how proteasomes are regulated to promote changes in the cyto architecture, size and survival of cells, and how this process affects age-related neuronal degeneration. We recently discovered a novel proteasome regulatory mechanism that offers unique opportunities to study proteasome regulation in the context of both normal organismal development, and in response to stress and aging. In particular, we found that the ADP-ribosyl transferase Tankyrase (TNKS) binds to and critically activates the proteasome regulator PI31 (Proteasome Inhibitor of 31kDa) to promote 26S proteasome assembly. These results suggest a potential mechanistic link between energy metabolism, NAD+, DNA-damage and proteasome regulation that is likely to play important roles in development, protein homeostasis and aging. Here, we will investigate the biological role and regulation of TNKS/PI31-mediated proteasome activation. We propose to use a multi-disciplinary approach that integrates Drosophila genetics, developmental biology, cell biology, and neurobiology, biochemistry, and small- molecule chemical inhibitors. Amongst other things, we will test the specific hypotheses that the TNKS/PI31-pathway is regulated by NAD+ that activation of this pathway protects against phototoxic stress and that diminished activity of this pathway with age causes increased vulnerability to neuronal degeneration. The current proposal brings, for the first time, the full power of Drosophila genetics and molecular biology to investigate these questions and combines it with biochemical studies in both insect and mammalian cells to explore new paths towards the clinic. We expect that this project will fundamentally advance our understanding of how protein degradation is regulated and provide new insights how to manipulate this process for the treatment of human diseases.