Proteases, cystic fibrosis and the epithelial sodium channel (ENaC).

Proteases perform a diverse array of biological functions. From simple peptide digestion for nutrient absorption to complex signaling cascades, proteases are found in organisms from prokaryotes to humans. In the human airway, proteases are associated with the regulation of the airway surface liquid layer, tissue remodeling, host defense and pathogenic infection and inflammation. A number of proteases are released in the airways under both physiological and pathophysiological states by both the host and invading pathogens. In airway diseases such as cystic fibrosis, proteases have been shown to be associated with increased morbidity and airway disease progression. In this review, we focus on the regulation of proteases and discuss specifically those proteases found in human airways. Attention then shifts to the epithelial sodium channel (ENaC), which is regulated by proteolytic cleavage and that is considered to be an important component of cystic fibrosis disease. Finally, we discuss bacterial proteases, in particular, those of the most prevalent bacterial pathogen found in cystic fibrosis, Pseudomonas aeruginosa.

Which patients are seen by an occupational psychiatry service?

BACKGROUND: Common mental disorders are the leading cause of sickness absence but are frequently misdiagnosed and undertreated. It is against this background that a specialist occupational psychiatry clinic was established at a London teaching hospital. AIMS: To explore the nature of patients and complaints seen in the clinic and investigate whether this form of service provision reached patients who may have otherwise been missed in the gap between primary and secondary care. METHODS: We reviewed the case notes of 51 consecutive new clinic assessments using a data extraction form, gathering information on socio-demographic and occupational details; the nature, duration and severity of symptoms [as assessed by Health of the Nation Outcome Scale (HoNOS)]; diagnosis; prior treatment and the outcome of the clinic appointment. RESULTS: Only half of those seen in the new clinic were currently on sick leave. The most common diagnosis was depression with most having symptoms lasting longer than 9 months. Sixty-five per cent had a medium or high HoNOS rating. Although 75% had received treatment from their general practitioner, the majority remained functionally impaired, and only 31% had been seen in secondary care. CONCLUSIONS: Specialist occupational psychiatry clinics do not replicate the work already being done by standard mental health services. Patients referred to a new specialist clinic within an occupational health department had chronic, debilitating psychiatric illnesses, which in many cases had failed to respond adequately to primary care treatment and were at risk of falling into the gap between primary and secondary services.

A novel paradigm for rapid ABT-737-induced apoptosis involving outer mitochondrial membrane rupture in primary leukemia and lymphoma cells.

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.

Breast implant associated anaplastic large cell lymphoma: The UK experience. Recommendations on its management and implications for informed consent.

BACKGROUND: Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is a rare, Non-Hodgkin lymphoma arising in the capsule of breast implants. BIA-ALCL presents as a recurrent effusion and/or mass. Tumours exhibit CD30 expression and are negative for Anaplastic Lymphoma Kinase (ALK). We report the multi-disciplinary management of the UK series and how the stage of disease may be used to stratify treatment. METHODS: Between 2012 and 2016, 23 cases of BIA-ALCL were diagnosed in 15 regional centres throughout the UK. Data on breast implant surgeries, clinical features, treatment and follow-up were available for 18 patients. RESULTS: The mean lead-time from initial implant insertion to diagnosis was 10 years (range: 3-16). All cases were observed in patients with textured breast implants or expanders. Fifteen patients with breast implants presented with stage I disease (capsule confined), and were treated with implant removal and capsulectomy. One patient received adjuvant chest-wall radiotherapy. Three patients presented with extra-capsular masses (stage IIA). In addition to explantation, capsulectomy and excision of the mass, all patients received neo-/adjuvant chemotherapy with CHOP as first line. One patient progressed on CHOP but achieved pathological complete response (pCR) with Brentuximab Vedotin. After a mean follow-up of 23 months (range: 1-56) all patients reported here remain disease-free. DISCUSSION: BIA-ALCL is a rare neoplasm with a good prognosis. Our data support the recommendation that stage I disease be managed with surgery alone. Adjuvant chemotherapy may be required for more invasive disease and our experience has shown the efficacy of Brentuximab as a second line treatment.

High CIP2A levels correlate with an antiapoptotic phenotype that can be overcome by targeting BCL-XL in chronic myeloid leukemia.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a predictive biomarker of disease progression in many malignancies, including imatinib-treated chronic myeloid leukemia (CML). Although high CIP2A levels correlate with disease progression in CML, the underlying molecular mechanisms remain elusive. In a screen of diagnostic chronic phase samples from patients with high and low CIP2A protein levels, high CIP2A levels correlate with an antiapoptotic phenotype, characterized by downregulation of proapoptotic BCL-2 family members, including BIM, PUMA and HRK, and upregulation of the antiapoptotic protein BCL-XL. These results suggest that the poor prognosis of patients with high CIP2A levels is due to an antiapoptotic phenotype. Disrupting this antiapoptotic phenotype by inhibition of BCL-XL via RNA interference or A-1331852, a novel, potent and BCL-XL-selective inhibitor, resulted in extensive apoptosis either alone or in combination with imatinib, dasatinib or nilotinib, both in cell lines and in primary CD34(+) cells from patients with high levels of CIP2A. These results demonstrate that BCL-XL is the major antiapoptotic survival protein and may be a novel therapeutic target in CML.

XIAP inhibition of caspase-3 preserves its association with the Apaf-1 apoptosome and prevents CD95- and Bax-induced apoptosis.

Ligation of death receptors or formation of the Apaf-1 apoptosome results in the activation of caspases and execution of apoptosis. We recently demonstrated that X-linked inhibitor-of-apoptosis protein (XIAP) associates with the apoptosome in vitro. By utilizing XIAP mutants, we now report that XIAP binds to the 'native' apoptosome complex via a specific interaction with the small p12 subunit of processed caspase-9. Indeed, we provide the first direct evidence that XIAP can simultaneously bind active caspases-9 and -3 within the same complex and that inhibition of caspase-3 by the Linker-BIR2 domain prevents disruption of BIR3-caspase-9 interactions. Recent studies suggest that inhibition of caspase-3 is dispensable for its anti-apoptotic effects. However, we clearly demonstrate that inhibition of caspase-3 is required to inhibit CD95 (Fas/Apo-1)-mediated apoptosis, whereas inhibition of either caspase-9 or caspase-3 prevents Bax-induced cell death. Finally, we illustrate for the first time that XIAP mutants, which are incapable of binding to caspases-9 and -3 are completely devoid of anti-apoptotic activity. Thus, XIAP's capacity to maintain inhibition of caspase-9 within the Apaf-1 apoptosome is influenced by its ability to simultaneously inhibit active caspase-3, and depending upon the apoptotic stimulus, inhibition of caspase-9 or 3 is essential for XIAP's anti-apoptotic activity.

The transrepression arm of glucocorticoid receptor signaling is protective in mutant huntingtin-mediated neurodegeneration.

The unfolded protein response (UPR) occurs following the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and orchestrates an intricate balance between its prosurvival and apoptotic arms to restore cellular homeostasis and integrity. However, in certain neurodegenerative diseases, the apoptotic arm of the UPR is enhanced, resulting in excessive neuronal cell death and disease progression, both of which can be overcome by modulating the UPR. Here, we describe a novel crosstalk between glucocorticoid receptor signaling and the apoptotic arm of the UPR, thus highlighting the potential of glucocorticoid therapy in treating neurodegenerative diseases. Several glucocorticoids, but not mineralocorticoids, selectively antagonize ER stress-induced apoptosis in a manner that is downstream of and/or independent of the conventional UPR pathways. Using GRT10, a novel selective pharmacological modulator of glucocorticoid signaling, we describe the importance of the transrepression arm of the glucocorticoid signaling pathway in protection against ER stress-induced apoptosis. Furthermore, we also observe the protective effects of glucocorticoids in vivo in a Drosophila model of Huntington's disease (HD), wherein treatment with different glucocorticoids diminished rhabdomere loss and conferred neuroprotection. Finally, we find that growth differentiation factor 15 has an important role downstream of glucocorticoid signaling in antagonizing ER stress-induced apoptosis in cells, as well as in preventing HD-mediated neurodegeneration in flies. Thus, our studies demonstrate that this novel crosstalk has the potential to be effectively exploited in alleviating several neurodegenerative disorders.

Comparisons of the effects of tamoxifen, toremifene and raloxifene on enzyme induction and gene expression in the ovariectomised rat uterus.

This study compares the actions of oestradiol, tamoxifen, toremifene and raloxifene on enzyme and gene expression in uterine tissues of ovariectomised rats over 72 h. The time-course for the induction of ornithine decarboxylase by the compounds showed a rapid biphasic response, while for creatine kinase brain type (BB) there was a continued increase over 72 h. The efficacy of induction showed that, with both markers, oestradiol gave the highest induction level, followed by tamoxifen or toremifene and then raloxifene. RT-PCR demonstrated that all compounds decreased oestrogen receptor (ER) alpha, ERbeta and ERbeta2 gene expression, 8-24 h after the first dose, suggesting that down-regulation of ER is not the primary cause of the difference in efficacy between these compounds. Using cDNA arrays, expression of 512 genes was examined in the uteri of oestradiol- or tamoxifen-treated rats. Both compounds resulted in the up-regulation of heat-shock protein 27, telomerase-associated protein 1 and secretin. However, most surprising was the marked down-regulation of Wilms' tumour and retinoblastoma genes. We speculate that this may result in a loss of regulation of the transition from the G1 to the S phase in the cell cycle and may make cells more vulnerable to the carcinogenic effects of tamoxifen in this tissue.

A novel role for carboxylesterase in the elevation of cellular cysteine by esters of cysteine.

Esters of cysteine, such as cysteine isopropylester (CIPE) or cysteine cyclohexylester (CCHE), are efficient delivery systems for cysteine to cells. After enzymic cleavage, the esters of cysteine provide a source of cellular cysteine, which may support reduced glutathione (GSH) synthesis and/or act as a direct chemoprotectant. Reducing esterase activity of rat lung slices or isolated hepatocytes with paraoxon or bis(4-nitrophenyl) phosphate or by reducing the temperature to 4 degrees dramatically altered the metabolism of esters of cysteine; the initial increase in cellular cysteine was slowed, the residency time of cysteine esters in the extracellular pool was prolonged without substantially enhancing the levels of intracellular ester. Incubation of lung slices with CIPE at 4 degrees led to a marked increase in cellular cysteine, which prior inhibition of esterase activity abolished. Inhibiting the neutral amino acid uptake systems, ASC and L, while effecting the uptake of cysteine, did not reduce the elevation of cellular cysteine by CIPE. We propose that the elevation of cellular cysteine by esters of cysteine may be mediated by membrane associated esterase activity.

Cysteine isopropylester protects against paracetamol-induced toxicity.

Cysteine isopropylester (CIPE), a novel ester of cysteine, has been synthesized in order to evaluate its potential as a chemoprotectant. The increased lipophilicity of the ester relative to cysteine should facilitate its entry into cells where, following hydrolysis, it should act as an intracellular source of cysteine or be utilized for the synthesis of glutathione so protecting the cell against various types of chemical insult. In this study, we evaluate the ability of CIPE to protect against paracetamol-induced hepatotoxicity in mice. When administered to mice, CIPE produced a rapid but transient elevation of levels of non-protein sulphydryls (NPSH) in liver, lung, kidney and spleen. The greatest increase in NPSH was seen in the lung, but after 60 min all NPSH values had returned to control levels, demonstrating the capacity of the mouse to rapidly metabolize both CIPE and cysteine. In mice pretreated with benzo(a)pyrene, CIPE protected against paracetamol-induced toxicity as measured by the prevention of mortality, the fall in hepatic NPSH and the decreased elevation of serum transaminases. CIPE (1.5 mmol/kg) appeared as effective as N-acetylcysteine (1.5 mmol/kg). Higher doses of CIPE (3.0 mmol/kg) alone were toxic to mice induced with benzo(a)pyrene but not to control or phenobarbitone-induced mice. The mechanism of this increased toxicity is unclear. CIPE has a short in vivo half life but was capable of protecting against paracetamol-induced toxicity. The potential of CIPE and other related cysteine esters to act as chemoprotectants merits further investigation.

Elevation of cysteine and replenishment of glutathione in rat lung slices by cysteine isopropylester and other cysteine precursors.

In this study, we have used a rat lung slice model to compare the ability to several potential cysteine delivery systems (L-cysteine isopropylester, L-cysteine cyclohexylester, N-acetylcysteine, L,2-oxo-4-thiazolidine carboxylic acid and cysteine) to elevate cysteine and glutathione (GSH) levels in control lung slices and slices depleted of their GSH by diethyl maleate. The esters of cysteine produced the greatest rise in lung slice cysteine. All the cysteine delivery systems were capable of replenishing GSH in lung slices previously depleted of GSH by diethyl maleate.

Structure-activity relationships of cysteine esters and their effects on thiol levels in rat lung in vitro.

Pretreatment with cysteine esters increases cysteine (CySH) levels in rat lung and protects against the lethal effects of inhaled perfluoroisobutene in vivo. There are marked differences in the duration of protection achieved with different cysteine esters. In this study we have compared the uptake and metabolism of CySH, N-acetyl cysteine (NAc), cysteine esters and cystine esters in vitro using rat lung and liver homogenates and lung slices. Liver homogenates metabolized CySH and cysteine esters faster than lung homogenates. The half life (T1/2) of CySH in lung was 58.8 +/- 17.3 min and in liver was 14.0 +/- 1.6 min (mean +/- SEM). T1/2 of the esters in lung ranged between 6.5 and 12.1 min and in liver between 1.9 and 5.3 min. Cysteine tertiary butyl ester, which does not protect in vivo, was not hydrolysed to CySH by lung or liver homogenates. All esters increased and prolonged intracellular CySH concentrations in lung slices to a much greater extent than CySH itself. NAc did not raise intracellular CySH above that of the controls and no NAc appeared within the slice. After CySH incubation intracellular CySH was 0.9 +/- 0.1 nmol/mg wet wt at 10 min whereas after incubation with the esters it ranged between 2.60 and 3.65 nmol/mg wet wt. Cysteine cyclohexyl ester prolonged the increase of CySH the longest and cysteine methyl ester the shortest. CySH levels with cysteine cyclohexyl ester were 2.74 +/- 0.15 and 4.13 +/- 0.37 nmol/mg wet wt at 10 and 60 min, respectively, whereas with cysteine methyl ester, CySH levels were 2.60 +/- 0.5 and 1.25 +/- 0.08 nmol/mg wet wt at similar times. Cystine esters increased intracellular concentrations of both cystine and CySH. CySH concentrations ranged between 2.92 and 3.19 nmol/mg wet wt and cystine between 1.39 and 1.47 nmol/mg wet wt at 60 min. The elevation and duration of CySH in lung slices is well correlated with the duration of protection against perfluoroisobutene achieved in vivo.

Cytochemical localization of adenylate cyclase in cultured renal epithelial (A6) cells.

To characterize the vasopressin-adenylate cyclase (AC) signaling pathway in control of Na+ reabsorption in cultured renal (A6) cells, we determined the distribution of AC with a cytochemical technique using 5'-adenylylimidodiphosphate as substrate and cerium chloride as capturing agent. The addition of forskolin to the medium to stimulate AC activity increased the production of reaction deposits at the enzyme sites. To ensure that the cells were close to their physiological states, cytochemical reactions were performed on unfixed tissues. Subsequent postfixation adequately preserved the morphological features of the cells. AC was mainly restricted to the lateral folds of the cells while the apical membranes were devoid of any deposits. This result provided evidence that the V2-AC pathway is not present in the apical membrane and, hence, any vasopressin action on apical Na+ channels from the luminal side of the cell must involve other signaling pathways. The cytochemical results provided further morphological evidence of the functional coupling between the basolateral and apical membranes of renal cells. We examined the idea that highly variable basal rates of Na+ transport in young differentiating cell cultures may be related to the degree of AC activity. Cytochemical results apparently revealed highly variable amounts of deposits in these cells, but by quantitative analysis of AC activity we could find no significant differences between cells of 6, 14, and 21 days.

Customising an international disease management model to the needs of individual countries. Application to upper gastrointestinal disease.

The baseline economic model for upper gastrointestinal (UGI) disease was developed in the context of patterns of care and resource use within the UK. It provided the opportunity to evaluate the extent to which an economic model developed in one country could be applied to meet the pharmacoeconomic information needs of decision makers in another. The choice of countries for analysis was restricted to countries within the International Gastro Primary Care Group (IGPCG) who had previously agreed on the appropriateness of the basic clinical algorithm to their domestic healthcare environment. This provided a potential sample of 9 countries (Australia, Austria, Germany, Italy, The Netherlands, Sweden, Switzerland, the UK and the USA) of which the UK, Germany, Sweden and Switzerland were chosen as providing a broad spectrum of strategic and operating environments in which to test the international transferability of the economic model. The process and results obtained provide valuable evidence of the extent to which economic analyses can be transferred across national borders.

Development of an economic model for the management of upper gastrointestinal disease in primary care. Preliminary findings.

Health economic models for identifying therapeutic options that maximise health benefits from limited healthcare resources are being developed in a number of therapeutic areas. The development of such a model for upper gastrointestinal (UGI) symptoms to support decision-making by primary care clinicians is of particular importance, given the prevalence of this symptomatology. This economic model was based upon the clinical guidelines aimed at improving the management of UGI disorders at the primary care level that were developed by the International Gastro Primary Care Group. This paper discusses the derivation, methodology and results of the economic model developed to assess the resource implications arising from these clinical guidelines. In order to construct the economic model, it was necessary to identify the following: every therapeutic pathway followed by patients resource use along each pathway the probabilities of following alternative pathways. One crucial factor underlying the interpretation of results obtained from any economic model is the time period covered by the model. The model presented here analysed the initial 12-month treatment period of 'new' patients presenting with UGI symptoms. In order to test the implications of a longer term perspective, the model is currently being developed to analyse resource use over a 24-month period. The model demonstrates that utilising the predominant symptom approach to the diagnosis and treatment of patients with UGI disorders appears to provide significant benefits in terms of patient management and effective resource use. This factor, together with the more intensive use of Helicobacter pylori eradication therapy, provides the potential to reduce the cost of drugs for the treatment of UGI disorders by approximately 15% in the UK. A major strength of the model is its adaptability to a wide range of clinical and cost scenarios. Such adaptability enables the model to effectively reflect the potential resource implications in countries exhibiting significantly different levels of cost and patient management. In this manner, the model provides one valuable method by which clinicians can be supported in optimising the management of UGI disorders within current resource constraints.

A test of the structural model of initiation of dieting among adolescent girls.

This article reports alternative findings from a pilot study to those presented recently (Strong GF, Huon KG. J. Psychosom. Res., 1998; 44:315-326) in regard to the proposed model of sociopsychological processes involved in the initiation of dieting among young adolescent girls. One hundred thirteen female high school pupils completed a battery of questionnaires that assessed dieting status, dietary restraint, autonomous functioning, skill-related functioning, social influence, and family functioning. The results indicate that family functioning predicts dietary restraint but that this effect is mediated by peer influence to diet. Furthermore, family functioning was associated with autonomous functioning, suggesting that this relationship should be pursued in a future test of the model. This pattern of results is different from an earlier test of the model, which indicated only a parental influence on dieting status. The results confirm that peer influences should be retained as a causal factor in a reformulated structural model.

Changes in the Community Periodontal Index of Treatment Needs (CPITN) after periodontal treatment in a general dental practice.

The result of a study of the effectiveness of treatment of chronic periodontal disease in a general dental practice using CPITN and the time required to treat patients with differing CPITN scores indicated that although there were significant reductions in calculus and shallow pockets, the treatment did not eliminate positive CPITN scores; 41% scored 1, 24% scored 2, and 34% scored 3 or 4 after treatment. Very few patients had 'healthy' periodontal sextants at the first visit; the most frequent CPITN category was 3. Patients in this category took up most of the treatment time. The treatment times per patient were much lower than the estimates of the World Health Organisation for similar treatments.

Infective endocarditis and the dental practitioner: a review of 53 cases involving litigation.

OBJECTIVE: To review episodes of infective endocarditis involving dental procedures that have resulted in litigation and to determine if any clinical recommendations can be obtained. DESIGN: 13-year retrospective study. INTERVENTION: Patient records were analysed to identify the probable cause of infective endocarditis. All were judged to be caused by dental manipulations on the basis of dental procedure, cardiac pathology, infecting micro-organism and time between onset of infection and dental manipulation. MAIN OUTCOME MEASURES: Cases were analysed to check if appropriate national guidelines on antibiotic prophylaxis were followed. Status of patient dental records was also evaluated. RESULTS: Dental procedures implicated in infective endocarditis were exodontia (23), scaling (21), root canal therapy with extra-canal instrumentation (7) and minor oral surgery (2). No medical history was recorded in 10 patients. In a further 31 medical history was inadequate or out of date. Dentists involved with these cases failed to give prophylactic antibiotics (48), prescribed incorrect antibiotics (2), or gave antibiotics at inappropriate times (2). There was one episode of prophylaxis with amoxycillin failing despite it being given correctly. CONCLUSIONS: If litigation is to be avoided dental practitioners must keep accurate dental records, take an appropriate medical history that is kept up to date and adhere to national guidelines on antibiotic prophylaxis.

Evaluation and critical assessment of putative MCL-1 inhibitors.

High levels of BCL-2 family proteins are implicated in a failed/ineffective apoptotic programme, often resulting in diseases, including cancer. Owing to their potential as drug targets in cancer therapy, several inhibitors of BCL-2 family proteins have been developed. These primarily target specific members of the BCL-2 family, particularly BCL-2 and BCL-XL but are ineffective against MCL-1. Major efforts have been invested in developing inhibitors of MCL-1, which is commonly amplified in human tumours and associated with tumour relapse and chemoresistance. In this report, the specificity of several BCL-2 family inhibitors (ABT-263, UCB-1350883, apogossypol and BH3I-1) was investigated and compared with putative MCL-1 inhibitors designed to exhibit improved or selective binding affinities for MCL-1 (TW-37, BI97C1, BI97C10, BI112D1, compounds 6 and 7, and MCL-1 inhibitor molecule (MIM-1)). ABT-263, BI97C1, BI112D1, MIM-1 and TW-37 exhibited specificity in inducing apoptosis in a Bax/Bak- and caspase-9-dependent manner, whereas the other agents showed no killing activity, or little or no specificity. Of these inhibitors, only ABT-263 and UCB-1350883 induced apoptosis in a BCL-2- or BCL-XL-dependent system. In cells that depend on MCL-1 for survival, ABT-263 and TW-37 induced extensive apoptosis, suggesting that at high concentrations these inhibitors have the propensity to inhibit MCL-1 in a cellular context. TW-37 induced apoptosis, assessed by chromatin condensation, caspase processing and phosphatidylserine externalisation, in a BAK-dependent manner and in cells that require MCL-1 for survival. TW-37-mediated apoptosis was also partly dependent on NOXA, suggesting that derivatives of TW-37, if engineered to exhibit better selectivity and efficacy at low nanomolar concentrations, may provide useful lead compounds for further synthetic programmes. Expanded medicinal chemistry iteration, as performed for the ABT series, may likewise improve the potency and specificity of the evaluated MCL-1 inhibitors.

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1.

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.