Proteasome particle-rich structures are widely present in human epithelial neoplasms: correlative light, confocal and electron microscopy study

A novel cytoplasmic structure has been recently characterized by confocal and electron microscopy in H. pylori-infected human gastric epithelium, as an accumulation of barrel-like proteasome reactive particles colocalized with polyubiquitinated proteins, H. pylori toxins and the NOD1 receptor. This proteasome particle-rich cytoplasmic structure (PaCS), a sort of focal proteasome hyperplasia, was also detected in dysplastic cells and was found to be enriched in SHP2 and ERK proteins, known to play a role in H. pylori-mediated gastric carcinogenesis. However, no information is available on its occurrence in neoplastic growths. In this study, surgical specimens of gastric cancer and various other human epithelial neoplasms have been investigated for PaCSs by light, confocal and electron microscopy including correlative confocal and electron microscopy (CCEM). PaCSs were detected in gastric cohesive, pulmonary large cell and bronchioloalveolar, thyroid papillary, parotid gland, hepatocellular, ovarian serous papillary, uterine cervix and colon adenocarcinomas, as well as in pancreatic serous microcystic adenoma. H. pylori bodies, their virulence factors (VacA, CagA, urease, and outer membrane proteins) and the NOD1 bacterial proteoglycan receptor were selectively concentrated inside gastric cancer PaCSs, but not in PaCSs from other neoplasms which did, however, retain proteasome and polyubiquitinated proteins reactivity. No evidence of actual microbial infection was obtained in most PaCS-positive neoplasms, except for H. pylori in gastric cancer and capsulated bacteria in a colon cancer case. Particle lysis and loss of proteasome distinctive immunoreactivities were seen in some tumour cell PaCSs, possibly ending in sequestosomes or autophagic bodies. It is concluded that PaCSs are widely represented in human neoplasms and that both non-infectious and infectious factors activating the ubiquitin-proteasome system are likely to be involved in their origin. PaCS detection might help clarify the role of the ubiquitin-proteasome system in carcinogenesis.     

Polyubiquitinated proteins,proteasome, and glycogen characterize the particle-rich cytoplasmic structure (PaCS) of neoplastic and fetal cells

A particle-rich cytoplasmic structure (PaCS) concentrating ubiquitin–proteasome system (UPS) components and barrel-like particles in clear, cytoskeleton- and organelle-free areas has recently been described in some neoplasms and in genetic or infectious diseases at risk of neoplasia. Ultrastructurally similar particulate cytoplasmic structures, interpreted as glycogen deposits, have previously been reported in clear-cell neoplasms and some fetal tissues. It remains to be investigated whether the two structures are the same, colocalize UPS components and polysaccharides, and have a role in highly proliferative cells such as fetal and neoplastic cells. We used immunogold electron microscopy and confocal immunofluorescence microscopy to examine human and mouse fetal tissues and human neoplasms. Fetal and neoplastic cells both showed colocalization of polyubiquitinated proteins, 19S and 20S proteasomes, and polysaccharides, both glycogen and chondroitin sulfate, inside cytoplasmic structures showing all distinctive features of PaCSs. Poorly demarcated and/or hybrid (ribosomes admixed) UPS- and glycogen-enriched areas, likely stages in PaCS development, were also seen in some fetal cells, with special reference to those, like primary alveolar pulmonary cells or pancreatic centroacinar cells, having a crucial role in organogenesis. UPS- and glycogen-rich PaCSs developed extensively in clear-cell neoplasms of the kidney, ovary, pancreas, and other organs, as well as, in infantile, development-related tumors replicating fetal patterns, such as choroid plexus papilloma. UPS-mediated, ATP-dependent proteolysis and its potential energy source, glycogen metabolism, may have a crucial, synergic role in embryo-/organogenesis and carcinogenesis.     

In Vivo Accumulation of Helicobacter pylori Products,NOD1, Ubiquitinated Proteins and Proteasome in a Novel Cytoplasmic Structure

Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer), are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS) we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance.     

Lanosterol modulates proteostasis via dissolving cytosolic sequestosomes/aggresome-like induced structures

Sequestration of misfolded proteins into distinct cellular compartments plays a pivotal role in proteostasis and proteopathies. Cytoplasmic ubiquitinated proteins are sequestered by p62/SQSTM1 to deposit in sequestosomes or aggresome-like induced structures (ALIS). Most aggresome or ALIS regulators identified thus far are recruiters, while little is known about the disaggregases or dissolvers. In this research, we showed that lanosterol synthase and its enzymatic product lanosterol effectively reduced the number and/or size of sequestosomes/ALIS/aggresomes formed by endogenous proteins in the HeLa and HEK-293A cells cultured under both non-stressed and stressed conditions. Supplemented lanosterol did not affect the proteasome and autophagic activities, but released the trapped proteins from the p62-positive inclusions accompanied with the activation of HSF1 and up-regulation of various heat shock proteins. Our results suggested that the coordinated actions of disaggregation by lanosterol and refolding by heat shock proteins might facilitate the cells to recycle proteins from aggregates. The disaggregation activity of lanosterol was not shared by cholesterol, indicating that lanosterol possesses additional cellular functions in proteostasis regulation. Our findings highlight that besides protein modulators, the cells also possess endogenous low-molecular-weight compounds as efficient proteostasis regulators.     

Amyloid precursor protein accumulates in aggresomes in response to proteasome inhibitor

Aggresomes are cytoplasmic inclusions which are localized at the microtubule organizing center (MTOC) as a result of induced proteasome inhibition, stress or over-expression of certain proteins. Aggresomes are linked to the pathogenesis of many neurodegenerative diseases. Here we studied whether amyloid precursor protein (APP), a type-I transmembrane glycoprotein, is localized in aggresomes after exposure to stress condition. Using confocal microscopy we found that APP is located in aggresomes and co-localized with vimentin, γ-tubulin, 20S and ubiquitin at the MTOC in response to proteasome dysfunction. An interaction between vimentin and APP was found after proteasome inhibition suggesting that APP is an additional protein constituent of aggresomes. Suppression of the proteasome system in APP-HEK293 cells overexpressing APP or transfected with APP Swedish mutation caused an accumulation of stable, detergent-insoluble forms of APP containing poly-ubiquitinated proteins. In addition, brain homogenates from transgenic mice expressing human APP with the Arctic mutation demonstrated an interaction between APP and the aggresomal-marker vimentin. These data suggest that malfunctioning of the proteasome system caused by mutation or overexpression of pathological or non-pathological proteins may lead to the accumulation of stable aggresomes, perhaps contributing to the neurodegeneration.     

Scaled-down purification protocol to access proteomic analysis of 20S proteasome from human tissue samples: comparison of normal and tumor colorectal cells

The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the cytoplasmic 20S proteasome of adjacent normal and tumor colorectal cells arising from tissue samples of several patients. Proteomic analyses based on two-dimensional gel electrophoresis (2DE) and mass spectrometry showed that the subunit composition of 20S proteasomes from these normal and tumor cells were not significantly different. The proteasome activity was also assessed in the cytoplasmic extracts and was similar or higher in tumor colorectal than in the corresponding normal cells. The scaled-down 20S proteasome purification protocol developed here can be applied to any human clinical tissue samples and is compatible with further proteomic analyses.     

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.     

Proteomic characterization of aggregating proteins after the inhibition of the ubiquitin proteasome system

Protein aggregation, which is associated with the impairment of the ubiquitin proteasome system, is a hallmark of many neurodegenerative diseases. To better understand the contribution of proteasome inhibition in aggregation, we analyzed which proteins may potentially localize in chemically induced aggregates in human neuroblastoma tissue culture cells. We enriched for proteins in high-density structures by using a sucrose gradient in combination with stable isotope labeling with amino acids in cell culture (SILAC). The quantitative analysis allowed us to distinguish which proteins were specifically affected by the proteasome inhibition. We identified 642 potentially aggregating proteins, including the p62/sequestosome 1 and NBR1 ubiquitin-binding proteins involved in aggregation. We also identified the ubiquitin-associated protein 2 like (UBAP2L). We verified that it cofractionated with ubiquitin in the high-density fraction and that it was colocalized in the ubiquitin-containing aggregates after proteasome inhibition. In addition, we identified several chaperone proteins and used data from protein interaction networks to show that they potentially interact with distinct subgroups of proteins within the aggregating structures. Several other proteins associated with neurodegenerative diseases, like UCHL1, were identified, further underlining the potential of our analysis to better understand the aggregation process and proteotoxic stress caused by proteasome inhibition.     

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.     

Proteasome inhibition induces TNFR1 shedding from human airway epithelial (NCI-H292) cells

The type 1 55-kDa TNF receptor (TNFR1) is an important modulator of lung inflammation. Here, we hypothesized that the proteasome might regulate TNFR1 shedding from human airway epithelial cells. Treatment of NCI-H292 human airway epithelial cells for 2 h with the specific proteasome inhibitor clasto-lactacystin beta-lactone induced the shedding of proteolytically cleaved TNFR1 ectodomains. Clasto-lactacystin beta-lactone also induced soluble TNFR1 (sTNFR1) release from the A549 pulmonary epithelial cell line, as well as from primary cultures of human small airway epithelial cells and human umbilical vein endothelial cells. Furthermore, sTNFR1 release induced by clasto-lactacystin beta-lactone was not a consequence of apoptosis or the extracellular release of TNFR1 exosome-like vesicles. The clasto-lactacystin beta-lactone-induced increase in TNFR1 shedding was associated with reductions in cell surface receptors and intracytoplasmic TNFR1 stores that were primarily localized to vesicular structures. As expected, the broad-spectrum zinc metalloprotease inhibitor TNF-alpha protease inhibitor 2 (TAPI-2) attenuated clasto-lactacystin beta-lactone-mediated TNFR1 shedding, which is consistent with its ability to inhibit the zinc metalloprotease-catalyzed cleavage of TNFR1 ectodomains. TAPI-2 also reduced TNFR1 on the cell surface and attenuated the clasto-lactacystin beta-lactone-induced reduction of intracytoplasmic TNFR1 vesicles. This suggests that TNFR1 shedding induced by clasto-lactacystin beta-lactone involves the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. Together, these data are consistent with the conclusion that proteasomal activity negatively regulates TNFR1 shedding from human airway epithelial cells, thus identifying previously unrecognized roles for the proteasome and zinc metalloproteases in modulating the generation of sTNFRs.     

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging,aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.     

ALIS are Stress-Induced Protein Storage Compartments for Substrates of the Proteasome and Autophagy

Misfolded proteins can be directed into cytoplasmic aggregates such as aggresomes and dendritic cellaggresome-like induced structures (DALIS). DALIS were originally identified in lipopolysaccharidestimulateddendritic cells and act as storage compartments for polyubiquitinated Defective RibosomalProducts (DRiPs) prior to their clearance by the proteasome. Here we demonstrate that ubiquitinatedprotein aggregates that are similar to DALIS, and not related to aggresomes, can be observed in severalcell types in response to stress, including oxidative stress, transfection, and starvation. Significantly, bothimmune and non-immune cells could form these aggresome-like induced structures (ALIS). Proteinsynthesis was essential for ALIS formation in response to oxidative stress, indicating that DRiP formationwas required. Furthermore, puromycin, which increases DRiP formation, was sufficient to induce ALISformation. Inhibition of either proteasomes or of autophagy interfered with ALIS clearance in puromycintreated cells. Autophagy inhibition enhanced ALIS formation under a variety of stress conditions. Duringstarvation, ALIS formation in autophagy-deficient cells was only partially inhibited by protein synthesisinhibitors, indicating that both long-lived proteins and DRiPs can be targeted to ALIS. Together, thesefindings demonstrate that ALIS act as generalized stress-induced protein storage compartments forsubstrates of the proteasome and autophagy.     

The importance of nuclear import in protection of the vitamin D receptor from polyubiquitination and proteasome‐mediated degradation

Others and we previously showed that the vitamin D receptor (VDR) is subject to degradation by the 26S proteasome and that treatment with 1,25‐dihydroxyvitamin D₃ (1,25D₃) inhibited this degradation. In the present study, we found that in osteoblasts, but not in intestinal epithelial cells, the VDR was susceptible to degradation by the 26S proteasome. The subcellular site for degradation of the VDR in osteoblasts is the cytoplasm and the site for ligand‐dependent protection of the VDR from the 26S proteasome is the chromatin. These direct relationships between nuclear localization and protection of the VDR from 26S proteasome degradation led us to hypothesize that the unoccupied cytoplasmic VDR is a substrate for polyubiquitination, which targets VDR for degradation by the 26S proteasome, and that nuclear localization has the ability to protect the VDR from polyubiquitination and degradation. To test these hypotheses, we used Cos‐1 cells transfected with human VDR and histidine‐tagged ubiquitin expression vectors. We found that unoccupied VDR was polyubiquitinated and that 1,25D₃ inhibited this modification. Mutations in the nuclear localization signal of VDR (R49W/R50G and K53Q/R54G/K55E) or in the dimerization interface of VDR with retinoid X receptor (M383G/Q385A) abolished the ability of 1,25D₃ to protect the VDR from polyubiquitination, although these mutations had no effect on the ligand‐binding activity of VDR. Therefore, we concluded that in some cellular environments unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation and that ligand‐dependent heterodimerization and nuclear localization protect the VDR from these modifications. J. Cell. Biochem. 110: 926–934, 2010. © 2010 Wiley‐Liss, Inc.     

ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy

Misfolded proteins can be directed into cytoplasmic aggregates such as aggresomes and dendritic cell aggresome-like induced structures (DALIS). DALIS were originally identified in lipopolysaccharide-stimulated dendritic cells and act as storage compartments for polyubiquitinated Defective Ribosomal Products (DRiPs) prior to their clearance by the proteasome. Here we demonstrate that ubiquitinated protein aggregates that are similar to DALIS, and not related to aggresomes, can be observed in several cell types in response to stress, including oxidative stress, transfection, and starvation. Significantly, both immune and nonimmune cells could form these aggresome-like induced structures (ALIS). Protein synthesis was essential for ALIS formation in response to oxidative stress, indicating that DRiP formation was required. Furthermore, puromycin, which increases DRiP formation, was sufficient to induce ALIS formation. Inhibition of either proteasomes or of autophagy interfered with ALIS clearance in puromycin treated cells. Autophagy inhibition enhanced ALIS formation under a variety of stress conditions. During starvation, ALIS formation in autophagy-deficient cells was only partially inhibited by protein synthesis inhibitors, indicating that both long-lived proteins and DRiPs can be targeted to ALIS. Together, these findings demonstrate that ALIS act as generalized stress-induced protein storage compartments for substrates of the proteasome and autophagy.     

Proteasome inhibition induces <Emphasis Type="Italic">hsp30</Emphasis> and <Emphasis Type="Italic">hsp70</Emphasis> gene expression as well as the acquisition of thermotolerance in <Emphasis Type="Italic">Xenopus laevis</Emphasis> A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.     

Reversible cytoplasmic localization of the proteasome in quiescent yeast cells

The 26S proteasome is responsible for the controlled proteolysis of a vast number of proteins, including crucial cell cycle regulators. Accordingly, in Saccharomyces cerevisiae, 26S proteasome function is mandatory for cell cycle progression. In budding yeast, the 26S proteasome is assembled in the nucleus, where it is localized throughout the cell cycle. We report that upon cell entry into quiescence, proteasome subunits massively relocalize from the nucleus into motile cytoplasmic structures. We further demonstrate that these structures are proteasome cytoplasmic reservoirs that are rapidly mobilized upon exit from quiescence. Therefore, we have named these previously unknown structures proteasome storage granules (PSGs). Finally, we observe conserved formation and mobilization of these PSGs in the evolutionary distant yeast Schizosaccharomyces pombe. This conservation implies a broad significance for these proteasome reserves.     

Withaferin A Induces Proteasome Inhibition,Endoplasmic Reticulum Stress,the Heat Shock Response and Acquisition of Thermotolerance

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.     

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II--aging of nondividing cells.

Oxidized/cross-linked intracellular protein materials, known as ceroid pigment, age pigment, or lipofuscin, accumulate in postmitotic tissues. It is unclear, however, whether diminishing proteolytic capacities play a role in the accumulation of such oxidized intracellular proteins. Previous studies revealed that the proteasome is responsible for the degradation of most oxidized soluble cytoplasmic and nuclear proteins and, we propose, for the prevention of such damage accumulations. The present investigation was undertaken to test the changes in protein turnover, proteasome activity, lysosome activity, and protein oxidation status during the aging of nondividing cells. Since the companion paper shows that both proteasome activity and the overall protein turnover decline during proliferative senescence whereas the accumulation of oxidized proteins increases significantly, we decided to use the same human BJ fibroblasts, this time at confluency, at different PD levels (including those that are essentially postmitotic) to investigate the same parameters under conditions where cells do not divide. We find that the activity of the cytosolic proteasome declines dramatically during senescence of nondividing BJ fibroblasts. The peptidyl-glutamyl-hydrolyzing activity was particularly affected. This decline in proteasome activity was accompanied by a decrease in the overall turnover of short-lived (radiolabeled) proteins in the nondividing BJ fibroblasts. On the other hand, no decrease in the actual cellular proteasome content, as judged by immunoblots, was found. The decline in the activity of the proteasome was also accompanied by an increased accumulation of oxidized proteins, especially of oxidized and cross-linked material. Unlike the loss of lysosomal function seen in our accompanying studies of proliferative senescence (1), however, the present study of hyperoxic senescence in nondividing cells actually revealed marked increases in lysosomal cathepsin activity in all but the very 'oldest' postmitotic cells. Our comparative studies of proliferating (1) and nonproliferating (this paper) human BJ fibroblasts reveal a good correlation between the accumulation of oxidized/cross-linked proteins and the decline in proteasome activity and overall cellular protein turnover during in vitro senescence, which may predict a causal relationship during actual cellular aging.     

Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.