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Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities.

ABSTRACT: Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ??? and the major subpopulation ? comprised proteasomes containing immuno-subunits ?1i and ?5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ?? contained only ?5i beside standard-subunits. In favour of a relative increase of the major subpopulation ?, subpopulation ?? and ??? were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.

SUBMITTER: Gohlke S

PROVIDER: S-EPMC3889881 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities.

Gohlke Sabrina S Mishto Michele M Textoris-Taube Kathrin K Keller Christin C Giannini Carolin C Vasuri Francesco F Capizzi Elisa E D'Errico-Grigioni Antonia A Kloetzel Peter-Michael PM Dahlmann Burkhardt B

Age (Dordrecht, Netherlands) 20130522 1

Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits β1i and β5i be ...[more]

PMID: 23690132

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Project description:1. On storage of rat liver mitochondria at 0 degrees , water content, total amino acid content and leakage of protein all rose steadily over a 72hr. period. The initial ratio of intramitochondrial to extramitochondrial amino acid concentration lay between 18 and 24. Initially this rose, but it then fell to 1.9 at the end of storage. The concentration gradient between internal and external amino acids was relatively constant throughout the period. These processes were accentuated at 22 degrees and 40 degrees , the concentration gradient reaching 70mumoles/ml., water content rising to 8.3mg./mg. dry wt. and protein leakage reaching 42% of total mitochondrial protein. ;Swelling agents' produced no correlated changes in amino acid production and swelling. 2. Added glutamate was not concentrated within the pellet of whole or disrupted mitochondria. Endogenous amino acids were distributed evenly between the pellet and the supernatant of disrupted mitochondria. It is concluded that amino acids are produced within mitochondria and that adsorption and uptake from the medium do not contribute significantly to amino acids in the pellet. 3. beta-Glycerophosphate, a lysosome protectant, increased amino acid production by rat liver mitochondria. Treatment with Triton X-100 and disruption by freezing and thawing showed that 56% of proteolytic activity was ;free' in whole mitochondria, whereas only 11% of acid phosphatase activity, a lysosomal enzyme, was ;free'. 4. ;Light' mitochondria contained 30% more neutral proteolytic activity but 300% more acid phosphatase activity than ;heavy' mitochondria. 5. Electron micrographs of mitochondrial preparations showed less than one particle in 500 that could be identified as a lysosome. Treatment with Triton X-100 disrupted the structure of roughly 50% of the mitochondria; the rest appeared to retain their membrane, cristae and ground substance. Freezing and thawing caused gross swelling and loss of ground substance and rupture of external membranes. 6. Of the recovered proteolytic activity, 81% at pH7.4 and 70% at pH5.8 were found in the high-speed supernatant of broken mitochondria. A further fivefold increase in specific activity was found in the first protein fraction obtained by Sephadex G-50 gel filtration. 7. Between 60 and 80% of proteolytic activity was found in the 40-60%-saturated ammonium sulphate precipitate. Almost all of the soluble-fraction proteolytic activity could be recovered in a pH5.0 supernatant. 8. The results give no support to the view that mitochondrial neutral proteolytic activity reflects lysosomal content. 9. The possible role of intramitochondrial amino acid production and the proteolysis of internal barriers in passive swelling of mitochondria is discussed.

Project description:Toxicokinetic and metabolism studies are considered crucial to dose setting and interpretation of data from rodent carcinogenicity studies. The metabolism and distribution studies are usually conducted in 6 to 8 week old rodents. However cancer studies generally involve exposures from 6 weeks of age to 108 weeks or more of age. It is recognized that age-related differences in sensitivities to some xenobiotics occur in several rodent models including the rat. However, the potential for changes in metabolism with age has received little attention in the toxicology community especially in regard to extrapolation of rodent data to human risk assessment. The advent of microarray technology has allowed a more global assessment of genes whose products are associated with xenobiotic metabolism. We studied hepatic gene expression in 38 untreated F344/N rats at approximately 32, 58 and 84 weeks of age corresponding to 6, 12, and 18 months on study. Statistical analysis of microarray (SAM) identified differential expression between the youngest and the oldest rats for genes whose products are involved in phase I and phase II metabolic pathways. Fifteen of 30 cytochrome P450 genes from CYP families 1 â 4 showed marked decrease in expression at 84 weeks while only two showed increased expression. The results of this study demonstrate prominent decreased expression in CYP family genes involved in xenobiotic metabolism in untreated rats as they age. The assumption of a constant metabolic profile when extrapolating results from long-term chemical exposures needs to be reconsidered. Experiment Overall Design: Animals and Study Design. Male Fischer 344 rats approximately 36 Â± 3 days old were supplied by Taconic laboratory animals (Germantown, NY) and were approximately six weeks old when placed in study rooms as sentinel animals for two-year studies. All studies were conducted at Battelle Science and Technology International (Columbus, Ohio). The livers from the male rat sentinels from three studies at six months (15 total rats), two studies at twelve months (10 total rats) and three studies at eighteen months (13 total rats) were used in this study. The sampling times corresponded to approximately 32, 58 and 84 weeks of age for the rats. The studies in which the sentinels were a part were approved by Battelleâs Institute Animal Care and Use Committee and were conducted in accordance with the guide for the care and use of laboratory animals (ILAR 1996). Details of the animal care and handling for studies conducted at Battelle have been reported previously (Boorman et al. 2005). Experiment Overall Design: The rats were kept in a cage until necropsy. The rats were anesthetized with CO2/O2, blood samples obtained for serology by cardiac puncture, the abdominal cavity was opened and the portal vein was severed. The left liver lobe was promptly removed and a cross section was placed in 10% neutral buffered formalin (NFB). The liver section was embedded, sectioned and stained with H&E and examined. The remainder of the left lobe was processed for subsequent RNA isolation. Experiment Overall Design: RNA isolation. The left hepatic lobe was cut into 0.5 cm cubes or smaller and immersed in RNALaterÂ® (Ambion, Austin, TX) within 4 minutes of necropsy. The tissues were stored in RNALaterÂ® overnight at 4+/-30C, then stored at - 20+/-10C until RNA isolation (within 60 days). Details of the RNA isolation procedures have been previous published (Boorman et al. 2005). Briefly, the RNA samples were frozen at - 700 C and shipped to the NTP repository until transfer to Paradigm Array Labs (Icoria, Inc., A Clinical Data Inc. Company, RTP, NC) for microarray analysis. RNA was isolated from individual rats. Equal amounts of RNA from sixty-three male sentinel rats (25 rats at 32 weeks of age, 25 rats at 58 weeks of age and 13 rats at 84 weeks of age) were used to form a composite control pool for comparison with the individual rats at each age. Experiment Overall Design: Microarray Hybridizations. One Âµg of total RNA from either an individual rat or from the pooled sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturerâs protocol. The amount and quality of the resulting fluorescently labeled cRNA was assessed using a Nanodrop ND-100 spectrophometer and an Agilent Bioanalyzer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the Agilent Rat Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17 hrs, prior to washing and scanning. Data was extracted from the resulting images using Agilentâs Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, CA).

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Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities.

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Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities.

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