Proteasome Expression Research Articles

The proteasome load versus capacity balance determines apoptotic sensitivity of multiple myeloma cells to proteasome inhibition

AbstractProteasome inhibitors (PIs) are effective against multiple myeloma (MM), but the mechanisms of action and bases of individual susceptibility remain unclear. Recent work linked PI sensitivity to protein synthesis and proteasome activity, raising the question whether different levels of proteasome expression and workload underlie PI sensitivity in MM cells (MMCs). Exploiting human MM lines characterized by differential PI sensitivity, we report that highly sensitive MMCs express lower proteasome levels and higher proteasomal workload than relatively PI-resistant MMCs, resulting in the accumulation of polyubiquitinated proteins at the expense of free ubiquitin (proteasome stress). Manipulating proteasome expression or workload alters apoptotic sensitivity to PI, demonstrating a cause-effect relationship between proteasome stress and apoptotic responses in MMCs. Intracellular immunostaining in primary, patient-derived MMCs reveals that polyubiquitinated proteins hallmark neoplastic plasma cells, in positive correlation with immunoglobulin (Ig) content, both intra- and interpatient. Moreover, overall proteasome activity of primary MMCs inversely correlates with apoptotic sensitivity to PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMCs to PI, potentially providing a framework for identifying indicators of responsiveness and designing novel combination therapies.

Disruption of Rpn4-induced proteasome expression in Saccharomyces cerevisiae reduces cell viability under stressed conditions.

The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription activator Rpn4 upregulates the proteasome genes and is rapidly degraded by the assembled proteasome. Previous studies have shown that rpn4Delta cells are sensitive to a variety of stresses. However, the contribution of the loss of Rpn4-induced proteasome expression to the rpn4Delta phenotypes remains unclear because Rpn4 controls numerous genes other than the proteasome genes. Here we construct a yeast strain in which one of the essential proteasome genes, PRE1, is no longer induced by Rpn4. We show that the active proteasome level is lower in this strain than in the wild-type counterpart. Moreover, we demonstrate that loss of Rpn4-induced proteasome expression leads to cell-cycle delay in G2/M and sensitizes cells to various stresses. To our knowledge, this is the first report that explicitly reveals the physiological function of Rpn4-induced proteasome expression. This study also provides a tool for understanding the interactions between proteasome homeostasis and other cellular processes.

Proteasome Stress Causes Apoptotic Sensitivity of Multiple Myeloma Cells to Proteasome Inhibition

Proteasome inhibitors (PI) proved to be extremely effective against different types of cancer, particularly against Multiple Myeloma (MM), a frequent and still incurable plasma cell malignancy. Phase II clinical trials showed that more than 50% of MM patients fail to respond to bortezomib, the only PI currently approved for clinical use. However, the mechanisms of action and bases of individual susceptibility to PI remain largely unclear, with no reliable predictor of response identified so far. Recent evidences linking proteasome activity and Ig synthesis to susceptibility to PI suggest that the exquisite sensitivity of MM cells (MMC) to PI might be explained by an imbalance between the efficiency of the ubiquitin (Ub)-proteasome pathway and the demand for proteasome-mediated degradation. We set out to explore this hypothesis both in vitro and ex vivo. To achieve this aim, we employed human MM cell lines characterized by differential apoptotic sensitivity to PI (U266 and RPMI8226, fairly resistant cell lines, versus MM.1S, an extremely sensitive one) and primary, patient derived MMC. In MM cell lines, we found that high apoptotic sensitivity to PI is associated with lower expression of active proteasomes (as assessed by decreased expression of cleaved catalytic subunits and enzymatic assays with fluorogenic substrates in cell extracts), together with higher proteasomal workload (demonstrated by higher proteasome-dependent loss of TCA-insoluble radioactivity in pulse-chase assays). Indeed, MM.1S cells displayed 2–3 times lower proteasomal activity as compared to the more resistant U266 and RPMI8226 cells, both on a per cell basis and upon normalization by protein content. Together with the reduced proteasome capacity, MM.1S cells showed a consistently higher production of client proteins for the Ub-proteasome pathway. Such an increased load appears to be the consequence of a higher production of Rapidly Degraded Polypeptides (RDP). These are newly synthesized proteins which are quickly redirected to proteasome-mediated degradation. The imbalance between proteasomal load and capacity results in remarkable accumulation of poly-Ub proteins at the expense of free Ub (as established by both western blotting and immunofluorescence), unveiling basal proteasome stress in PI-sensitive MMC. In order to establish a causal link between proteasome stress and sensitivity to PI, we pharmacologically modulated either proteasome expression or workload and successfully altered PI-induced apoptosis. As predicted, increasing proteasome workload by means of ER stressors (e.g. tunicamycin, thapsigargin, brefeldin A) dramatically enhances susceptibility to PI, while a raise in proteasomal activity (achieved by exploiting the proteasome stress response, an adaptive mechanism by which mammalian cells induce proteasome biogenesis in response to either decreased proteasome function or increased proteasomal demand), confers marked resistance to PI-induced apoptosis. Having established cause-effect relationships between determinants of proteasome stress and vulnerability to PI in vitro, we then asked if our model could be used to predict responsiveness to PI in MM patients. In keeping with this hypothesis, intracellular immunostaining in primary, patient-derived MMC reveals that accumulation of poly-Ub proteins specifically hallmarks neoplastic plasma cells, indicating that the cancer compartment in MM patients suffers from proteasome stress. Moreover, poly-Ub levels positively correlate with Ig content, both intra- and inter-patient, suggesting a direct effect of Ig synthesis and/or retention on proteasome functional load. Finally, overall proteasome activity of primary MMC inversely correlates with the intrinsic apoptotic sensitivity to PI as assessed ex vivo, providing a rationale for the assessment of this parameter as a potential predictor of the in vivo response to bortezomib or other PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMC to PI, providing both a novel predictive tool of potential prognostic value and the framework for novel combination therapies aimed at exacerbating proteasome stress in MM.

Multiple Forms of Proteasomes and their Role in Tumor Fate

Mammalian and human cells contain multiple forms of proteasomes different in their structure and functions. 26S-proteasome pool regulates most cellular processes through ATP- and ubiquitin-dependent hydrolysis of proteins participating in these processes. This function is possible due to 19S-subparticle capable to recognize ubiquitinated proteins, unfold and direct them into the proteolytic chamber. 20S-proteasome pool is capable to degrade some damaged and foreign proteins in an ATP- and ubiquitin-independent manner. Among both proteasome pools, the immune proteasomes effectively produce antigen epitopes for MHC class I molecules and play a crucial role in the antitumor immunity. Excluding the immune proteasomes from their cells, numerous tumors avoid the immune system. On the contrary, tumor cells enhance the expression of the housekeeping proteasomes and 19S-subparticle. At this time, bortezomib, a patented proteasome inhibitor, is used as an anticancer therapy. This drug induces the cell cycle arrest and apoptosis of dividing tumor cells. However, bortezomib causes, firstly, deviations in immune functions, secondly, the increase of the housekeeping proteasome expression by a feedback mechanism. Taking into account the recent patents, we consider prospects of the development of new drugs directed to the regulation of the expression of the immune and housekeeping proteasomes and 19S-subparticle in tumor cells.

Partial proteasome inhibition in human fibroblasts triggers accelerated M1 senescence or M2 crisis depending on p53 and Rb status

Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression are reduced during aging and replicative senescence. Its activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs), whereas partial proteasome inhibition triggers an irreversible premature senescent state in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence (RS and PS, respectively), in this study we investigated their implication in proteasome inhibition-mediated PS. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we reveal that proteasome activity inhibition to levels normally found in senescent human cells results in immediate growth arrest and/or moderate increase of apoptotic death. These effects are independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated PS can only be initiated and maintained in the presence of functional p53. More specifically, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the continuous interplay between the integrity of proteasome function, senescence and cell survival.

Structure and properties of transcriptional networks driving selenite stress response in yeasts

BackgroundStress responses provide valuable models for deciphering the transcriptional networks controlling the adaptation of the cell to its environment. We analyzed the transcriptome response of yeast to toxic concentrations of selenite. We used gene network mapping tools to identify functional pathways and transcription factors involved in this response. We then used chromatin immunoprecipitation and knock-out experiments to investigate the role of some of these regulators and the regulatory connections between them.ResultsSelenite rapidly activates a battery of transcriptional circuits, including iron deprivation, oxidative stress and protein degradation responses. The mRNA levels of several transcriptional regulators are themselves regulated. We demonstrate the existence of a positive transcriptional loop connecting the regulator of proteasome expression, Rpn4p, to the pleiotropic drug response factor, Pdr1p. We also provide evidence for the involvement of this regulatory module in the oxidative stress response controlled by the Yap1p transcription factor and its conservation in the pathogenic yeast C. glabrata. In addition, we show that the drug resistance regulator gene YRR1 and the iron homeostasis regulator gene AFT2 are both directly regulated by Yap1p.ConclusionThis work depicted a highly interconnected and complex transcriptional network involved in the adaptation of yeast genome expression to the presence of selenite in its chemical environment. It revealed the transcriptional regulation of PDR1 by Rpn4p, proposed a new role for the pleiotropic drug resistance network in stress response and demonstrated a direct regulatory connection between oxidative stress response and iron homeostasis.

Pattern of proteasome expression in Walker 256 tumor cells after their transplantation into the Brattleboro rats with genetic defect of vasopressin synthesis

Pattern of proteasome expression in Walker 256 tumor cells after their transplantation into the Brattleboro rats with genetic defect of vasopressin synthesis

Identification and Characterization of a Membrane Receptor for Proteolysis-Inducing Factor on Skeletal Muscle

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (K(d), in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (K(d), 1.1 x 10(-7) mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [(35)S]PIF to triton solublized membranes from C(2)C(12) cells provided evidence for a binding protein of apparent M(r) of approximately 40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target.

Bortezomib Has Anti-Proliferative and Apoptotic Effects Against CML Stem Cells, Including the Quiescent Population.

CML is a disease of the haemopoietic stem cell (HSC) caused by the constitutively active tyrosine kinase Bcr-Abl. The current first-line therapy for CML is imatinib (IM), a tyrosine kinase inhibitor (TKI) effective in establishing durable disease control in the majority of patients in chronic phase. However, the quiescent leukaemic HSC (Ph+HSC) population is insensitive to the apoptotic effects of this and more potent TKIs - such as dasatinib or nilotinib. These TKIs exert potent anti-proliferative effects against CML stem and progenitor cells, resulting in accumulation of quiescent cells in vitro. This Ph+HSC reservoir of disease in treated patients is now recognised to be a critical target if we wish to achieve complete eradication of residual disease. Bcr-Abl+ cells have abnormally high levels of proteasome activity and expression and in common with other cancer cells have been shown to be relatively more sensitive to the effects of proteasome inhibitors (PI) than their non-malignant counterparts. We have assessed the potential role for PI in CML using Bcr-Abl+ cell lines (K562 and Ba/F3 with mutant/wild type Bcr-Abl) and Ph+HSC obtained from leukapheresis products of patients, taken with consent, at time of diagnosis with CML. We have demonstrated apoptotic and anti-proliferative effects of PI (MG132 and bortezomib) in wild type and mutated Bcr-Abl+ cell lines (including T315I) and in Ph+HSC (n=7) by viable cell counts, thymidine incorporation and flow cytometry for annexin V and 7AAD. The apoptotic effects also occurred in the quiescent Ph+HSC fraction with an IC50 of 5–7.5nM (bortezomib)- similar to that seen in the total CD34+ cell population and lower than that previously reported for cells from multiple myeloma patients. These results were compared to parallel FACS-sorted cell populations from non-CML Ph- leukapheresis products and a therapeutic window was seen. CFSE tracking of cell division in CML samples in vitro indicated depletion of the quiescent fraction, in contrast to the accumulation of these cells demonstrated with IM, nilotinib or dasatinib, either alone or in combination. The intracellular effects of exposure of these cell populations to bortezomib were assessed by quantitatively measuring the intracellular accumulation of polyubiquitinated proteins seen following PI treatment. Interestingly no clear effect on Bcr-Abl activity was demonstrated with PI treatment of Ph+HSC (measured by immunoblotting with antibody to phosphorylated Crkl), though in Bcr-Abl+ cell lines detectable activity was reduced. Drug combination experiments (Calcusyn) were performed with bortezomib and IM or dasatinib to assess the effect of simultaneous inhibition of Bcr-Abl and the proteasome - the combination of a TKI with a PI resulted in additive effects, however no clear synergistic effects of significance were seen. Our results indicate that PI are capable of inducing CML stem cell specific apoptosis, suggesting the proteasome may be a relevant target for eradication of residual disease in CML following TKI therapy.

Proteasome activation during cardiac hypertrophy by the chaperone H11 Kinase/Hsp22

The regulation of protein degradation by the proteasome during cardiac hypertrophy remains largely unknown. Also, the proteasome translocates to the nuclear periphery in response to cellular stress in yeast, which remains unexplored in mammals. The purpose of this study was to determine the quantitative and qualitative adaptation of the proteasome during stable cardiac hypertrophy. We measured proteasome activity, expression and sub-cellular distribution in a model of chronic cardiac hypertrophy induced by the stress-response chaperone H11 Kinase/Hsp22 (Hsp22). Over-expression of Hsp22 in a transgenic (TG) mouse leads to a 30% increase in myocyte cross-sectional area compared to wild-type (WT) mice (P < 0.01). Characterization of the proteasome in hearts from TG mice vs. WT revealed an increased expression of both 19S and 20S subunits (P < 0.05), a doubling in 20S catalytic activity (P < 0.01), a redistribution of both subunits from the cytosol to the nuclear periphery, and a four-fold increase in nuclear-associated 20S catalytic activity (P < 0.001). The perinuclear proteasome co-localized and interacted with Hsp22. Inhibition of proteasome activity by epoxomicin reduced hypertrophy in TG by 50% (P < 0.05). Adeno-mediated over-expression of Hsp22 in isolated cardiac myocytes increased both cell growth and proteasome activity, and both were prevented upon inhibition of the proteasome. Similarly, stimulation of cardiac cell growth by pro-hypertrophic stimuli increased Hsp22 expression and proteasome activity, and proteasome inhibition in that setting prevented hypertrophy. Proteasome inhibitors also prevented the increase in rate of protein synthesis observed after over-expression of Hsp22 or upon addition of pro-hypertrophic stimuli. Hsp22-mediated cardiac hypertrophy promotes an increased expression and activity, and a subcellular redistribution of the proteasome. Inhibition of the proteasome reverses cardiac hypertrophy upon Hsp22 over-expression or upon stimulation by pro-hypertrophic hormones, and also blocks the stimulation of protein synthesis in these conditions.

Muscle wasting in diabetic and in tumor-bearing rats: Role of oxidative stress

Cachexia is a debilitating syndrome characterized by body weight loss, muscle wasting, and anemia. Muscle wasting results from an altered balance between protein synthesis and degradation rates. Reactive oxygen species are indicated as crucial players in the onset of muscle protein hypercatabolism by upregulating elements of the ubiquitin–proteasome pathway. The present study has been aimed at evaluating comparatively the involvement of oxidative stress in the pathogenesis of skeletal muscle wasting in two different experimental models: rats rendered hyperglycemic by treatment with streptozotocin and rats bearing the Yoshida AH-130 ascites hepatoma. For this purpose, both tumor bearers and diabetic animals have been treated with dehydroepiandrosterone (DHEA), a multifunctional steroid endowed with multitargeted antioxidant properties. We show that diabetic rats and AH-130 rats share several features, hypoinsulinemia, occurrence of oxidative stress, and positive response to DHEA administration, although the extent of the effects of DHEA largely differs between diabetic animals and tumor-bearing rats. The hypercatabolism, evaluated in terms of proteasome activity and expression of atrogin-1 and MuRF1, is activated in AH-130 rats, whereas it is lacking in streptozotocin-treated rats. Moreover, we demonstrate that the role of oxidative stress can interfere with muscle wasting through different mechanisms, not necessarily involving NF-κB activation. In conclusion, the present results show that, although skeletal muscle wasting occurs in both diabetic rats and tumor-host rats, the underlying mechanisms are different. Moreover, despite oxidative stress being detectable in both experimental models, its contribution to muscle wasting is not comparable.

Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants

The effects of cadmium (Cd) on cellular proteolytic responses were investigated in the roots and leaves of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 3 and 10 days in the presence of 0.3-300 microM Cd and compared to control plants grown in the absence of Cd. Roots of Cd treated plants accumulated four to fivefold Cd as much as mature leaves. Although 10 days of culture at high Cd concentrations inhibited plant growth, tomato plants recovered and were still able to grow again after Cd removal. Tomato roots and leaves are not modified in their proteolytic response with low Cd concentrations (< or =3 microM) in the incubation medium. At higher Cd concentration, protein oxidation state and protease activities are modified in roots and leaves although in different ways. The soluble protein content of leaves decreased and protein carbonylation level increased indicative of an oxidative stress. Conversely, protein content of roots increased from 30 to 50%, but the amount of oxidized proteins decreased by two to threefold. Proteolysis responded earlier in leaves than in root to Cd stress. Additionally, whereas cysteine- and metallo-endopeptidase activities, as well as proteasome chymotrypsin activity and subunit expression level, increased in roots and leaves, serine-endopeptidase activities increased only in leaves. This contrasted response between roots and leaves may reflect differences in Cd compartmentation and/or complexation, antioxidant responses and metabolic sensitivity to Cd between plant tissues. The up-regulation of the 20S proteasome gene expression and proteolytic activity argues in favor of the involvement of the 20S proteasome in the degradation of oxidized proteins in plants.

Role of increased expression of the proteasome in the protective effects of sulforaphane against hydrogen peroxide-mediated cytotoxicity in murine neuroblastoma cells

The 26S proteasome is responsible for degradation of abnormal proteins and may play a role in cell survival upon oxidative stress. The indirect antioxidant sulforaphane (SFN) protects animal tissues from chemical toxicants by increasing the expression of several families of Nrf2-regulated genes. The role of induction of the 26S proteasome in cytoprotection by SFN was investigated in murine neuroblastoma Neuro2A cells. SFN enhanced the expression of the catalytic subunits of the proteasome, as well as proteasomal peptidase activities in these cells. Such treatment with SFN protected cells from hydrogen peroxide-mediated cytotoxicity in a manner dependent on proteasomal function. Inhibition of proteasome activities using pharmacological interventions significantly attenuated the protective effects of SFN against hydrogen peroxide cytotoxicity, as well as protein oxidation. Moreover, overexpression of the catalytic subunit PSMB5 enhanced proteasome function and led to elevated resistance against hydrogen peroxide toxicity and extent of protein oxidation compared to blank-plasmid-transfected cells. Pretreatment of PSMB5-overexpressing cells with SFN did not further enhance this resistance. Collectively, these results suggest that the cytoprotective effects of SFN against oxidative stress are in part due to up-regulation of the proteasome system. Therefore, inducers of proteasome expression may ameliorate the accumulation of damaged proteins associated with neurodegeneration and other diseases in whose etiologies protein oxidation plays a role.

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin-like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S-20S-19S proteasomes, and abrogation of expression of beta 1i, beta 2i and beta 5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic beta1, beta2 and beta 5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase-like, trypsin-like and chymotrypsin-like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, gamma-irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti-apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro-apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis-resistant phenotype.

Tissue specific increase of the catalytic subunits of the 26S proteasome by indirect antioxidant dithiolethione in mice: Enhanced activity for degradation of abnormal protein

Decreases in the 26S proteasome are related to the toxicities of abnormal protein aggregates and may contribute to pathogenesis of degenerative diseases. Therefore, maintenance of proteasome function can be a novel strategy to protect cells against abnormal protein-mediated toxicity. In the present study, we have demonstrated the tissue specific increase of the catalytic subunits of the proteasome in mice following oral administration of 3 H-1,2-dithiole-3-thione (D3T, 0.5 mmol/kg), which functions as a cancer preventive agent in animal and human studies. Expression of the 20S catalytic core subunits PSMB5, PSMB6, and PSMB7 were increased in liver, lung, small intestine, and colon of mice at 24 h after D3T treatment. Elevated expression of proteasome catalytic subunits led to increases in proteasomal peptidase activities in these tissues. Oral administration of D3T also exerted a pharmacodynamic action in some brain regions of these mice and proteasomal peptidase activities were significantly elevated in the cerebral cortex–hippocampus. Moreover, tissue extracts from D3T-treated mice and cell lysates obtained from D3T-incubated murine neuroblastoma cells exhibited the enhanced capacity to degrade mutant human SOD1G93A protein. These results indicate that the catalytic subunits of the 26S proteasome are inducible in multiple tissues of mouse including brain by exogenous chemical treatment. Increased proteasome expression by inducers may have a role in protection/attenuation of protein aggregate-mediated disorders.

The neuroprotective effects of the Wlds gene are correlated with proteasome expression rather than apoptosis

The Wld(s) gene (slow Wallerian degeneration) specifically delays axonal degeneration following injury and in several models of neurodegenerative diseases. It thus provides an interesting tool to study mechanisms of neurodegeneration. We previously crossed the Wld(s) mice with a mouse mutant that has a motoneuron disease (pmn for progressive motor neuronopathy) and showed that the Wld(s) gene prevented axonal loss, increased the life-span and prolonged the survival of the motoneuron cell bodies. In this study we show that spinal motoneurons of pmn/pmn mice, as opposed to axons, die by apoptosis that cannot be prevented by the Wld(s) gene. However, this same gene could partially rescue the proteasome impairment observed in motoneuron cell bodies and axons of pmn/pmn mice. We conclude that the neuroprotective effect of the Wld(s) gene is not related to an inhibition of apoptosis but could possibly be linked to a regulation in proteasome expression.

Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase.

Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies in vitro have suggested that activation of the dsRNA-dependent protein kinase (PKR) may be responsible for these changes in protein synthesis and degradation. In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg−1) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2α phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-κB (NF-κB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia.

Diminished feedback regulation of proteasome expression and resistance to proteasome inhibitors in breast cancer cells

Clinical trials with proteasome inhibitor Bortezomib (also named Velcade or PS-341) has shown promising results for some cancers. However, other types of cancers including breast cancer do not respond well to Bortezomib. To understand the cause of the drug resistance, we compared the regulation of proteasome expression and the sensitivity to proteasome inhibitors between human breast cancer cells and nontumorigenic mammary epithelial cells. We found that, while the endogenous expression level is much higher, the potential of feedback expression in response to proteasome inhibitors is much lower in the breast cancer cells. Furthermore, the breast cancer cells are much more resistant to proteasome inhibitors compared to the nontumorigenic mammary epithelial cells. Biochemical analysis showed that the pathway of Bortezomib-induced apoptosis is apparently defective in the breast cancer cells. Together, these results provide an explanation for the inefficacy of Bortezomib in the clinical trials for breast cancer patients. The likelihood of combination therapy with Bortezomib and other anti-cancer agents for breast cancer is also discussed.

Skeletal Muscle Atrophy, a Link between Depression of Protein Synthesis and Increase in Degradation

Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2alpha). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2alpha phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRDelta6. Myotubes expressing PKRDelta6 showed no increase in eIF2alpha phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Inhibition of PKR prevented nuclear migration of NF-kappaB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-kappaB. Phosphorylation of PKR and eIF2alpha was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin–proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin–proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA 2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR.