MEDIATION OF CHEMOTHERAPY-INDUCED APOPTOSIS BY THE LYSOSOMAL PROTEASE CATHEPSIN D

One of the most common hallmarks of cancer is dysregulation of cellular apoptotic processes. A comprehensive knowledge of the underlying mechanisms of the apoptotic machinery is vital for the identification of new drug targets and the development of innovative agents that stimulate the cell death process in cancer cells. Studies have shown that the lysosomal protease cathepsin D is important in the extrinsic apoptotic pathway stimulated by the death receptor ligands for TNFR1 and FAS, as well as by oxidative stress and the protein kinase C inhibitor staurosporine. To date, the role of cathepsin D in the chemotherapy-induced apoptotic pathway has not been characterized. This project examined the role of the lysosomal protease cathepsin D in chemotherapy-induced apoptosis of HeLa and U937 cells. The data demonstrated that following stimulation of U937 cells with the chemotherapy drug VP-16, cathepsin D was released into the cytosol approximately 4 hours after drug treatment. This release was selective for cathepsin D, as cathepsin B and the lysosomal markers LAMP and â-hexosaminidase were not released into the cytosol following VP-16 treatment. Inhibitors of caspases andcathepsin D had no effect on cathepsin D release, demonstrating that cathepsin D release occurred independently of caspase and cathepsin D activities. Downregulation of cathepsin D expression in U937 and Hela cells using siRNA was found to inhibit cell death resulting from a variety of stimuli, including death receptor ligands, oxidative stress, PKC inhibitors, and importantly, chemotherapy drugs. In addition, U937 and HeLa cells expressing cathepsin D siRNA exhibited delayed cytochrome c release and caspase-3 activation following VP-16 treatment. Moreover, isolated mitochondria from wild-type U937 cells released cytochrome c in response to cytosolic extracts that were treated with cathepsin D, suggesting that cathepsin D acts on a cytosolic factor to induce cytochrome c release. Inhibition of caspases had no impact on cytochrome c release provoked by cathepsin D-cleaved cytosolic extract, demonstrating that caspases are not mediators of cathepsin D-induced cytochrome c release. Taken together, these results demonstrate that cathepsin D is an important component of the apoptotic pathway and that it acts via an intermediary cytosolic factor to promote cytochrome c release and caspase activation during chemotherapy-induced apoptosis

Membrane-Associated Conformation of HIV‑1 Nef Investigated with Hydrogen Exchange Mass Spectrometry at a Langmuir Monolayer

In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane

Subtle Dynamic Changes Accompany Hck Activation by HIV‑1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor

The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenyl­furano­pyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells

Antiretroviral Drug Discovery Targeting the HIV-1 Nef Virulence Factor

While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs

Molecular priming of Lyn by GPVI enables an immune receptor to adopt a hemostatic role

The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates