Decreased protein and puromycinyl-peptide degradation in livers of senescent mice.
Ontology highlight
ABSTRACT: Liver protein-degradation rates were determined in young and old C57B1 mice by the method of Swick & Ip [(1974) J. Biol. Chem. 249, 6836-6841]. The results indicated a marked age-related increase in the half-lives of short-lived proteins in the nuclear, mitochondrial, lysosomal and 100000 g-supernatant cellular fractions and in total trichloroacetic acid-precipitable proteins. The efficiency of the degradation system in removing aberrant proteins from livers of young and old mice was tested. The time required for 50% disappearance of puromycinyl-peptides changed from about 20 min in 6-month-old mice to approx. 150 min in 24-month-old animals. These findings suggest that in old animals the proteolytic activity involved in degradation of aberrant proteins, and presumably of "native proteins, is markedly defective.
Project description:Site-1 protease (S1P) cleaves membrane-bound sterol regulatory element-binding proteins (SREBPs), allowing their transcription-stimulating domains to translocate to the nucleus where they activate genes governing lipid synthesis. S1P is a potential target for lipid-lowering drugs, but the effect of S1P blockade in animals is unknown. Here, we disrupt the S1P gene in mice. Homozygous germ-line disruptions of S1P were embryonically lethal. To disrupt the gene inducibly in liver, we generated mice homozygous for a floxed S1P allele and heterozygous for a transgene encoding Cre recombinase under control of the IFN-inducible MX1 promoter. When IFN was produced, 70-90% of S1P alleles in liver were inactivated, and S1P mRNA and protein were reduced. Nuclear SREBPs declined, as did mRNAs for SREBP target genes. Cholesterol and fatty acid biosynthesis in hepatocytes declined by 75%. Low density lipoprotein (LDL) receptor mRNA declined by 50%, as did the clearance of (125)I-labeled LDL from plasma, but plasma cholesterol fell, suggesting that LDL production was reduced. These data raise the possibility that S1P inhibitors may be effective lipid-lowering agents, but they suggest that nearly complete inhibition will be required.
Project description:Ibuprofen, an inhibitor of prostanoid biosynthesis, is a common pharmacological agent used for the management of pain, inflammation and fever. However, the chronic use of ibuprofen at high doses is associated with increased risk for cardiovascular, renal, gastrointestinal and liver injuries. The underlying mechanisms of ibuprofen-mediated effects on liver remain unclear. To determine the mechanisms and signaling pathways affected by ibuprofen (100 mg/kg/day for seven days), we performed proteomic profiling of male mice liver with quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. More than 300 proteins were significantly altered between the control and ibuprofen-treated groups. The data suggests that several major pathways including (1) energy metabolism, (2) protein degradation, (3) fatty acid metabolism and (4) antioxidant system are altered in livers from ibuprofen treated mice. Independent validation of protein changes in energy metabolism and the antioxidant system was carried out by Western blotting and showed sex-related differences. Proteasome and immunoproteasome activity/expression assays showed ibuprofen induced gender-specific proteasome and immunoproteasome dysfunction in liver. The study observed multifactorial gender-specific ibuprofen-mediated effects on mice liver and suggests that males and females are affected differently by ibuprofen.
Project description:5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1-/- primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2'-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2'-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence.
Project description:The gallbladder normally lies within a fossa on the visceral surface of the liver. The primary purpose of this study was to determine whether the volume of this fossa was reduced after cholecystectomy. Livers were obtained from embalmed cadavers of 19 females and 15 males with a mean age of 84.1 ± 10.8 yrs. The presence of a gallbladder was assessed, the volume of the irregularly-shaped gallbladder fossa determined from a mold of the fossa, and the dimensions of each fossa were estimated. The mean volume of gallbladder fossae from livers with gallbladders (n = 26; 13 females and 13 males) was 31.01 ± 17.82 ml, which was significantly greater than fossae in livers without gallbladders (n = 8, 6 females, 2 males) which was 8.75 ± 4.72 ml (P<0.0001). This difference still was significant after correcting fossa volume for overall liver weight and length of the femur. Livers with gallbladders had significantly larger dimensions (depth, length, and width) of their fossae molds than did livers without gallbladders (P<0.05). The largest percentage difference between the two groups in these dimensions was in the fossae depth, and there was a significant, positive correlation between all three of these dimensions and the overall volume of the fossae. Even looking only at female livers which tend to be smaller, gallbladder fossa volume was reduced in livers without a gallbladder. Thus, the present study demonstrated that the mean gallbladder fossa volume was significantly decreased in livers lacking gallbladders, even after correcting for the liver weight and size of the individual. While the mechanisms behind these changes in fossa volume currently are unknown, alterations in mechanical pressure relayed to adjacent liver cells after gallbladder removal may play a role in these fossa volume differences.
Project description:Aging inducing the development of senescent cells (SNCs) in various tissues is considered as the main cause of the age-related diseases. Senotherapy has become a promising anti-aging therapy. However, the effectivity and side-effect of senolytic agents are still concern. Here, we observed the downregulation of senescence-related genes by adoptive infusion of natural killer (NK) cells in 26 cases in peripheral blood CD3+ T cells. NK cell treatment also significantly decreased levels of senescence markers and senescence-associated secretory phenotypes (SASPs) in three senescent adipose tissues when culturing them together. Interestingly, cytotoxic activity of mouse NK cells against SNCs was significantly enhanced by dopamine in vitro through D1-like receptors. Acein, dopamine-releasing peptide, promoted the adoptive infusion of NK cells in effectively eliminating SNCs in a variety of tissues and reduced local and systemic SASPs in aging mice but Acein alone did not have the senolytic effect. These data demonstrated that adoptive infusion of NK cells is an effective means in removing SNCs, and peptide Acein combined with NK cells further enhances this effect in aging mice.
Project description:NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.
Project description:The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3delta but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery.
Project description:Although aging in the liver contributes to the development of chronic liver diseases such as NAFLD and insulin resistance, little is known about the molecular and metabolic details of aging in hepatic cells. To examine these issues, we used sequential oxidative stress with hydrogen peroxide to induce premature senescence in AML12 hepatic cells. The senescent cells exhibited molecular and metabolic signatures, increased SA-βGal and γH2A.X staining, and elevated senescence and pro-inflammatory gene expression that resembled livers from aged mice. Metabolic phenotyping showed fuel switching towards glycolysis and mitochondrial glutamine oxidation as well as impaired energy production. The senescent AML12 cells also had increased mTOR signaling and decreased autophagy which likely contributed to the fuel switching from β-oxidation that occurred in normal AML12 cells. Additionally, senescence-associated secretory phenotype (SASP) proteins from conditioned media of senescent cells sensitized normal AML12 cells to palmitate-induced toxicity, a known pathological effect of hepatic aging. In summary, we have generated senescent AML12 cells which displayed the molecular hallmarks of aging and also exhibited the aberrant metabolic phenotype, mitochondrial function, and cell signaling that occur in the aged liver.
Project description:MicroRNAs are short, non-coding RNA sequences that regulate genes at the post-transcriptional level and have been shown to be important in development, tissue differentiation, and disease. Limited attention has been given to the natural variation in miRNA expression across genetically diverse populations even though it is well established that genetic polymorphisms can have a profound effect on mRNA levels. Expression level of 577 miRNAs in the livers of 70 strains of inbred mice was assessed, and we found that miRNA expression is highly stable across different strains. Globally, the expression of miRNA target transcripts does not correlate with miRNA expression, primarily due to the low variance of miRNA but high variance of mRNA expression across strains. Our results show that there is little genetic effect on the baseline miRNA levels in murine liver. The stability of mouse liver miRNA expression in a genetically diverse population suggests that treatment-induced disruptions in liver miRNA expression, a phenomenon established for a large number of toxicants, may indicate an important mechanism for the disturbance of normal liver function, and may prove to be a useful genetic background-independent biomarker of toxicant effect.
Project description:Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that bind and recruit an E3 ubiquitin ligase to a targeted protein of interest, often through the utilization of a small molecule inhibitor. To expand the possible range of kinase targets that can be degraded by PROTACs, we sought to develop a PROTAC utilizing a hydrocarbon-stapled peptide as the targeting agent to bind the surface of a target protein of interest. In this study, we describe the development of a proteolysis-targeting chimera, dubbed Stapled Inhibitor Peptide - PROTAC or StIP-TAC, linking a hydrocarbon-stapled peptide with an E3 ligase ligand for targeted degradation of Protein Kinase A (PKA). This StIP-TAC molecule stimulated E3-mediated protein degradation of PKA, and this effect could be reversed by the addition of the proteasomal inhibitor MG-132. Further, StIP-TAC treatment led to a significant reduction in PKA substrate phosphorylation. Since many protein targets of interest lack structural features that make them amenable to small molecule targeting, development of StIP-TACs may broaden the potential range of protein targets using a PROTAC-mediated proteasomal degradation approach.