The proteasome activator PA200 regulates tumor cell responsiveness to glutamine and resistance to ionizing radiation.

The cellular response to ionizing radiation (IR) involves a variety of mechanisms to repair damage and maintain cell survival. We previously reported that the proteasome activator PA200 promotes long-term cell survival after IR exposure. The molecular function of PA200 is to enhance proteasome-mediated cleavage after glutamate; however, it is not known how this molecular function promotes survival after IR exposure. Here, we report that upon IR exposure, cellular demand for exogenous glutamine is increased. Cells containing PA200 are capable of surviving this IR-induced glutamine demand, whereas PA200-deficient cells show impaired long-term survival. Additional glutamine supplementation reverses the radiosensitivity of PA200-knockdown cells suggesting impaired glutamine homeostasis in these cells. Indeed, PA200-knockdown cells are unable to maintain intracellular glutamine levels. Furthermore, when extracellular glutamine is limiting, cells that contain PA200 respond by slowing growth, but PA200-knockdown cells and cells in which post-glutamyl proteasome activity is inhibited are nonresponsive and continue rapid growth. This cellular unresponsiveness to nutrient depletion is also reflected at the level of the mTOR substrate ribosomal S6 kinase (S6K). Thus, inability to restrict growth causes PA200-deficient cells to continue growing and eventually die due to lack of available glutamine. Together, these data indicate an important role for PA200 and post-glutamyl proteasome activity in maintaining glutamine homeostasis, which appears to be especially important for long-term survival of tumor cells after radiation exposure.

Distinct molecular mechanisms responsible for bortezomib-induced death of therapy-resistant versus -sensitive B-NHL cells.

Resistance to currently available therapies is a major impediment to the successful treatment of hematological malignancies. Here, we used a model of therapy-resistant B-cell non Hodgkin lymphoma (B-NHL) developed in our laboratory along with primary B-NHL cells to study basic mechanisms of bortezomib activity. In resistant cells and a subset of primary B-NHLs, bortezomib treatment led to stabilization of Bak and subsequent Bak-dependent activation of apoptosis. In contrast to sensitive cells that die strictly by apoptosis, bortezomib was capable of killing resistant cells through activation of apoptosis or caspase-independent mechanism(s) when caspases were pharmacologically inhibited. Our data demonstrate that bortezomib is capable of killing B-NHL cells via multiple mechanisms, regardless of their basal apoptotic potential, and contributes to growing evidence that proteasome inhibitors can act via modulation of B-cell lymphoma 2 (Bcl-2) family proteins. The capacity of bortezomib to act independently of the intrinsic apoptotic threshold of a given B-NHL cell suggests that bortezomib-based therapies could potentially overcome resistance and result in relevant clinical activity in a relapsed/refractory setting.

What is the role of alternate splicing in antigen presentation by major histocompatibility complex class I molecules?

The expression of major histocompatibility complex (MHC) class I molecules on the cell surface is critical for recognition by cytotoxic T lymphocytes (CTL). This recognition event leads to destruction of cells displaying MHC class I-viral peptide complexes or cells displaying MHC class I-mutant peptide complexes. Before they can be transported to the cell surface, MHC class I molecules must associate with their peptide ligand in the endoplasmic reticulum (ER) of the cell. Within the ER, numerous proteins assist in the appropriate assembly and folding of MHC class I molecules. These include the heterodimeric transporter associated with antigen processing (TAP1 and TAP2), the heterodimeric chaperone-oxidoreductase complex of tapasin and ERp57 and the general ER chaperones calreticulin and calnexin. Each of these accessory proteins has a well-defined role in antigen presentation by MHC class I molecules. However, alternate splice forms of MHC class I heavy chains, TAP and tapasin, have been reported suggesting additional complexity to the picture of antigen presentation. Here, we review the importance of these different accessory proteins and the progress in our understanding of alternate splicing in antigen presentation.

Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability.

Proteasome activator PA200 enhances proteasome-mediated cleavage after acidic residues in vitro; however, its role within cells is not known. Here, we show that, in response to ionizing radiation, PA200 forms hybrid proteasomes with 19S caps and 20S core proteasomes that accumulate on chromatin, leading to an increase in proteolytic activity. Unlike many other proteins that respond to DNA damage, the response of PA200 appears to be independent of Ataxia Telangiectasia Mutated and p53, but dependent on DNA-dependent protein kinase activity. Nonetheless, PA200 is critical because PA200-knockdown cells show genomic instability and reduced survival after exposure to ionizing radiation. This phenotype is reproduced by specific inhibition of postglutamyl activity of proteasomes, but combined treatment with PA200 siRNA and postglutamyl inhibitor does not show additive effects on survival. Together, these data suggest a unique role for PA200 in genomic stability that is likely mediated through its ability to enhance postglutamyl cleavage by proteasomes.

Proteasomes and proteasome activator 200 kDa (PA200) accumulate on chromatin in response to ionizing radiation.

Proteasome activator 200 kDa (PA200) forms nuclear foci after exposure of cells to ionizing radiation and enhances proteasome activity in vitro. Within cells, it is unclear whether PA200 responds to radiation alone or in association with proteasomes. In the present study, we identified three forms of cellular PA200 (termed PA200i, ii and iii) at the mRNA and protein levels. Neither PA200ii nor PA200iii appears to associate with proteasomes. All detectable PA200i is associated with proteasomes, which indicates that PA200i and proteasomes function together within the cell. Consistent with this idea, we find that exposure of cells to radiation leads to an equivalent accumulation of both PA200i and core proteasomes on chromatin. This increase in PA200 and proteasomes on chromatin is not specific to the stage of cell cycle arrest since it occurs in cells that arrest in G(2)/M and cells that arrest in G(1)/S after exposure to radiation. These data provide evidence that PA200 and proteasomes function together within cells and respond to a specific radiation-induced damage independent of the stage of cell cycle arrest.