Pathway choice between proteasomal and autophagic degradation

Efficient degradation of abnormal or aggregated proteins is crucial to protect the cell against proteotoxic stress. Selective targeting and disposal of such proteins usually occurs in a ubiquitin-dependent manner by proteasomes and macroautophagy/autophagy. Whereas proteasomes are efficient in degrading abnormal soluble proteins, protein aggregates are typically targeted for degradation by autophagic vesicles. Both processes require ubiquitin-binding receptors, which are targeted to proteasomes via ubiquitin-like domains or to phagophores (the precursors to autophagosomes) via Atg8/LC3 binding motifs, respectively. The use of substrate modification by ubiquitin in both pathways raised the question of how degradative pathway choice is achieved. In contrast to previous models, proposing different types of ubiquitin linkages for substrate targeting, we find that pathway choice is a late event largely determined by the oligomeric state of the receptors. Monomeric proteasome receptors bind soluble substrates more efficiently due to their higher affinity for ubiquitin. Upon substrate aggregation, autophagy receptors with lower ubiquitin binding affinity gain the upper hand due to higher avidity achieved by receptor bundling. Thus, our work suggests that ubiquitination is a shared signal of an adaptive protein quality control system, which targets substrates for the optimal proteolytic pathway.     

Changes in DNA distributions and ploidy of cho cells as a function of time in culture

Summary Chinese hamster ovary (CHO) cells maintained in continuous culture for 3 to 5 months may undergo subtle changes in drug sensitivity response, growth kinetics, plating efficiencies, et cetera. Our studies done independently in two different laboratories, using flow cytometry, indicate that the DNA histogram patterns change at about 11 wk, from populations with an approximate diploid DNA content to populations also composed of triploid and tetraploid cells. Chromosome counts also change from distributions of 21 to 22 to populations of cells having 21 to 22, 34, to 35 and 44 to 46 chromosomes. These alterations occur earlier (at 8 to 9 wk) in cell populations previously treated with anticancer drugs. The research was supported by NIH Grant DHEW 5 RO1 CA 15397 and funds from Upjohn Co. Kalamazoo, MI.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer

The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.     

DNA ploidy and pS2 protein expression in breast cancer

The DNA content of ductal breast carcinomas of varying histological grade was measured using static image cytometry and correlated with pS₂ expression in the tumour cells. Our study was performed on imprint of surgical biopsies of 60 women with ductal breast cancer. A statistically significant difference was observed between pS₂⁺ expression and grade of malignancy ( P <0.001). The percentage of euploid tumours significantly decreased from grade I to grade II to grade III ( P =0.01). The percentage of aneuploid tumours increased from pS₂⁺ to pS₂⁻ breast tumours ( P <0.001). These findings may be indicative of pS₂ and DNA ploidy alterations and tumour aggressiveness.     

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.     

Src-dependent autophagic degradation of Ret in FAK-signalling-defective cancer cells

We have recently described that autophagic targeting of Src maintains cancer cell viability when FAK signalling is defective. Here, we show that the Ret tyrosine kinase is also degraded by autophagy in cancer cells with altered/reduced FAK signalling, preventing its binding to FAK at integrin adhesions. Inhibition of autophagy restores Ret localization to focal adhesions. Importantly, Src kinase activity is required to target Ret to autophagosomes and enhance Ret degradation. Src is thus a general mediator of selective autophagic targeting of adhesion-linked kinases, and Ret a second FAK-binding tyrosine kinase degraded through autophagy in cancer cells under adhesion stress. Src-by controlling not only its own degradation but also that of other FAK-binding partners-allows cancer cell survival, suggesting a new therapeutic strategy.     

Ultracytochemical localization of H＋—adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

H^-adenosine triphosphatase (H^ -ATPase) activity was demonstrated cytochemically in autophagic vacuoles(AVs) of rat hepatocytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase(p-NPPase) activity[1].When an inhibitor of H^ -ATPase,N-ethylmaleimide (NEM) or 4,4‘-diisothiocyanostilbene-2,2‘disulfonic acid,disodium salt (DIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H^ -ATPase.Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate(VBL).The number of AVs increased remarkably in hepatocytes from 40 min after VBL treatment.H^ -ATPase activity was observed mainly on the membranes of lysosomes and AVs.However,early forms of AVs containing only incompletely digested material showed no H^ -ATPase activity.Most AVs revealing a positive reaction seemed to be in advanced stages of development.Acid phosphatase acticity was demonstrable in mature but not in early forms of AVs.The present investigation showed that membranes of advanced stage AVs possess an H^ -ATPase which may be derived from lysosomal membranes.     

DNA ploidy and vimentin expression in primary breast cancer

DNA ploidy and vimentin expression in primary breast cancer
The DNA content of 50 breast cancers of varying tumour type, grade and stage was measured using static image cytometry, and correlated with vimentin expression in the tumour cells. A tendency to increased vimentin expression and aneuploidy was observed in high grade and late stage tumours¹. A statistically significant difference was observed in DNA index and ploidy balance between grade 1 and grades 2 and 3 carcinomas ( P <0.05) and between stage I and stage II carcinomas ( P <0.05). There was a significant difference in the expression of vimentin between grades 1, 2, 3 ( P <0,001), and stages I, II and III ductal carcinomas ( P <0.05). No significant difference was observed in the proliferation index and the degree of hyperploidy ( P >0.05). Clonal heterogeneity was observed in 25% of breast carcinomas, and was associated with increased vimentin expression. These changes may be indicative of genomic alteration and tumour aggressiveness.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.     

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death.     

Comparative metabolism and toxicity of chemical carcinogens in primary cultures of hepatocytes

Summary Hepatocytes were prepared by an in situ or biopsy perfusion of liver with collagenase. Hepatocytes from adult liver were cultured without serum on collagen-coated dishes in culture medium supplemented with hormones. Stable monolayers were established within 24 h and were maintained for up to 10 d. The hormone supplement maintained cytochrome P-450, a critical component of mixed function oxygenase responsible for activation of many procarcinogens. The addition of serum and phenobarbital to the cultures also maintained higher levels of mixed function oxygenase activity. Viable cultures were prepared from mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Metabolism studies revealed the rate of metabolism and the extent of covalent binding to macromolecules, including DNA. Measures of cytotoxicity and genotoxicity in vitro provide an indication of hepatonecrotic and hepatocarcinogenic potency in vivo. Comparative metabolism, cytotoxicity, and geonotoxicity studies provide a means of facilitating the extrapolation of toxicity data from laboratory animals to humans. Predoctoral trainee, Kathleen K. Dougherty, was supported by NIEHS training grant PHS ES07059-03     

PRKAA/AMPK restricts HBV replication through promotion of autophagic degradation

Adenosine monophosphate-activated protein kinase (AMPK) is a crucial energy sensor that maintains cellular energy homeostasis. AMPK plays a critical role in macroautophagy/autophagy, and autophagy facilitates hepatitis B virus (HBV) replication. To date, the intrinsic link among AMPK, autophagy and HBV production remains to be elucidated. Here, we demonstrate that PRKAA (a catalytic subunit of AMPK) is activated in response to HBV-induced oxidative stress, which in turn decreases the production of HBV. Mechanistic studies reveal that the autophagy machinery is associated with the inhibitory effect of PRKAA/AMPK on HBV production. Activation of PRKAA/AMPK promotes autolysosome-dependent degradation through stimulation of cellular ATP levels, which then leads to the depletion of autophagic vacuoles. Taken together, our data suggest that the activation of AMPK might be a stress response of host cells to restrict virus production through promotion of autophagic degradation. These findings therefore indicate that AMPK could provide a potential therapeutic target for HBV infection.     

Stress-induced polyubiquitination of proteasomal ubiquitin receptors targets the proteolytic complex for autophagic degradation

Ubiquitin (Ub) is a small protein (8 kDa) found in all eukaryotic cells, which is conjugated covalently to numerous proteins, tagging them for recognition by a downstream effector. One of the best characterized functions of Ub is targeting proteins for either selective degradation by the proteasome, or for bulk degradation by the autophagy-lysosome system. The executing arm of the UPS is the 26S proteasome, a large multicatalytic complex. While much is known about the synthesis and assembly of the proteasome's subunits, the mechanism(s) underlying its removal has remained obscure, similar to that of many other components of the ubiquitin-proteasome system. Our recent study identified autophagy as the degrading mechanism for the mammalian proteasome, mostly under stress conditions. Amino acid starvation induces specific ubiquitination of certain 19S proteasomal subunits that is essential for its binding to SQSTM1/p62, the protein that shuttles the ubiquitinated proteasome to the autophagic machinery. SQSTM1 delivers ubiquitinated substrates for proteasomal degradation via interaction of its PB1 domain with the 19S proteasomal subunit PSMD4/Rpn10, in situations where the proteasome serves as a “predator." In contrast, we found that the UBA domain of SQSTM1 is essential for its interaction with the ubiquitinated proteasome and its delivery to the autophagosome, rendering the proteasome a “prey.”     

A method for the comparative study of replicative DNA synthesis in GGT-positive and GGT-negative hepatocytes in primary culture isolated from carcinogen-treated rats

Summary The presence of gamma-glutamyl transpeptidase (GGT) in focal nodules of hepatocytes is a commonly used marker for the identification of preneoplastic cell populations. Female Fischer 344 rats were initiated with a single intragastric administration of 200 mg diethylnitrosamine/kg, altered cells were selected after 0.02% 2-acetylaminofluorene was given in the diet; this was followed by a partial hepatectomy and promotion with dietary sodium phenobarbital for 4 wk. A mixed-cell population of GGT-positive and GGT-negative hepatocytes was obtained after collagenase perfusion and Percoll purification. An enriched population of GGT-positive hepatocytes was obtained by a modified “panning” technique. With quantitative scintillation spectrometry and autoradiography of [³H]thymidine incorporation, replicative DNA synthesis of GGT-positive and GGT-negative rat hepatocytes was observed in both the mixed-cell population and the enriched GGT-positive and GGT-negative cell populations. Under the culture conditions used, GGT-positive cells showed a higher level of replicative DNA synthesis than did GGT-negative cells; this indicates that such altered hepatocytes in the stage of promotion possess an inherently greater capacity for all replication, as previously suggested from studies in vivo.     

Metalloproteinases 2 and -9 activity during promotion and progression stages of rat liver carcinogenesis

Activity of metalloproteinases 2 and 9 (MMP-2 and 9) during promotion and progression of rat liver carcinogenesis was investigated in a modified resistant hepatocyte model. Development of preneoplastic liver lesions positive for glutathione S-transferase 7-7-(GST-P 7-7-positive PNL) and tumors besides hepatocytes positive for proliferating cell nuclear antigen (PCNA) were quantified and compared to MMP-2 and-9 activity using gelatin zymography. Marked increases in GST-P 7-7-positive PNL development, PCNA labeling indices, MMP-2 (pro, intermediate and active forms) and pro-MMP-9 activity were observed after proliferative stimulus induced by 2-acetylaminofluorene (2-AAF) exposure cycles. After 2-AAF withdrawal, increase in MMP-2 activity was detected only in neoplastic mixed lesions, whereas active MMP-9 was increased in both PLN and neoplastic tissues. Our findings suggest that MMP-2 may be associated with proliferative events induced by 2-AAF rather than with selective growth of PNL and that MMP-9 could be associated with progression of PNL and neoplastic mixed lesions.     

MTOR-independent,autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1^G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1^G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1^G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1^G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1^G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.