Endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells: implications for Alzheimer's disease.

Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore cellular homeostasis via transcriptional and post-transcriptional mechanisms. ER stress is also reported to activate the ER overload response (EOR), which activates transcription via NF-κB. We previously demonstrated that UPR activation is an early event in pre-tangle neurons in Alzheimer's disease (AD) brain. Misfolded and unfolded proteins are degraded via the ubiquitin proteasome system (UPS) or autophagy. UPR activation is found in AD neurons displaying both early UPS pathology and autophagic pathology. Here we investigate whether activation of the UPR and/or EOR is employed to enhance the proteolytic capacity of neuronal cells. Expression of the immunoproteasome subunits ß2i and ß5i is increased in AD brain. However, expression of the proteasome subunits is not increased by the UPR or EOR. UPR activation does not relocalize the proteasome or increase overall proteasome activity. Therefore proteasomal degradation is not increased by ER stress. In contrast, UPR activation enhances autophagy and LC3 levels are increased in neurons displaying UPR activation in AD brain. Our data suggest that autophagy is the major degradational pathway following UPR activation in neuronal cells and indicate a connection between UPR activation and autophagic pathology in AD brain.

Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework for clinical trials.

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique ß-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.

Characterization of the ubiquitin-proteasome system in bortezomib-adapted cells.

Resistance towards the proteasome inhibitor bortezomib is poorly understood. We adapted the HL-60, ARH-77 and AMO-1 cell lines (myeloid leukemia, plasmocytoid lymphoma, myeloma) to bortezomib exceeding therapeutic plasma levels, and compared characteristics of the ubiquitin-proteasome system, alternative proteases and the unfolded protein response (UPR) between adapted cells and parental lines. Adapted cells showed increased transcription rates, activities and polypeptide levels of the bortezomib-sensitive beta5, but also of the beta2 proteasome subunit and consistently retained elevated levels of active beta1/beta5-type proteasome subunits in the presence of therapeutic levels of bortezomib. Bortezomib-adapted HL-60 cells showed increased expression and proteasome association of the 11S proteasome activator, and did not accumulate poly-ubiquitinated protein, activate the UPR or UPR-mediated apoptosis in response to bortezomib. The rate of protein biosynthesis was reduced, and the transcription of chaperone genes downmodulated. We did not observe major changes in the activities of TPPII, cathepsins or deubiquitinating proteases. We conclude that different types of bortezomib-adapted cell lines, including myeloma, show similar patterns of changes in the proteasomal machinery which result in residual proteasome activity in the presence of bortezomib and a quantitative balance between protein biosynthesis and destruction.

Activity patterns of proteasome subunits reflect bortezomib sensitivity of hematologic malignancies and are variable in primary human leukemia cells.

Proteasomal proteolysis relies on the activity of six catalytically active proteasomal subunits (beta1, beta2, beta5, beta1i, beta2i and beta5i). Applying a functional proteomics approach, we used a recently developed activity-based, cell-permeable proteasome-specific probe that for the first time allows differential visualization of individual active proteasomal subunits in intact primary cells. In primary leukemia samples, we observed remarkable variability in the amounts of active beta1/1i-, beta2/2i- and beta5/5i-type of subunits, contrasting with their constant protein expression. Bortezomib inhibited beta5- and beta1-type, but to a lesser extend beta2-type of subunits in live primary cells in vitro and in vivo. When we adapted the bortezomib-sensitive human acute myeloid leukemia cell line HL-60 to bortezomib 40 nM (HL-60a), proteasomal activity profiling revealed an upregulation of active subunits, and residual beta1/beta5-type of activity could be visualized in the presence of bortezomib 20 nM, in contrast to control cells. In a panel of cell lines from hematologic malignancies, the ratio between beta2-type and (beta1 + beta5)-type of active proteasomal polypeptides mirrored different degrees of bortezomib sensitivity. We thus conclude that the proteasomal activity profile varies in primary leukemia cells, and that the pattern of proteasomal subunit activity influences the sensitivity of hematologic malignancies toward bortezomib.