Post-Translational Modification of Cellular Proteins by Ubiquitin and Ubiquitin-Like Molecules: Role in Cellular Senescence and Aging

Grillari, Johannes; Grillari‐Voglauer, Regina; Jansen‐Dürr, Pidder

doi:10.1007/978-1-4419-7002-2_13

Cited by 31 publications

(22 citation statements)

References 246 publications

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“…Proteasome activity was found to be decreased in cellular senescence and organismal aging 7,8,9,10 . In the field of aging research, the focus is mainly on the removal of oxidized proteins where the proteasome plays a key role, and there is no conclusive evidence that protein ubiquitylation precedes degradation in this case 2 .…”

Section: Discussionmentioning

confidence: 99%

“…The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4,6 . Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7,8,9,10 . Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein.…”

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confidence: 99%

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Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Greussing¹,

Unterluggauer²,

Kozieł³

et al. 2012

JoVE

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Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1,2 . Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3 , cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4,6 . Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7,8,9,10 . Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein.To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)-dgn fusion protein (GFP-dgn, unstable) and a variant carrying a frameshift mutation (GFP-dgnFS, stable 11 ) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP-dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry. Video LinkThe video component of this article can be found at http://www.jove.com/video/3327/ Protocol 1. Plasmid Construction 1. Order custom oligo-nucleotides encoding for dgn (ACKNWFSSLSHFVIHL 11 ) and for dgnFS (HARTGSLACPTSSSICE) and ligate it into the pEGFP-C1 vector to obtain the fusion of the GFP with dgn/dgnFS (Figure 1). 2. Amplify the coding sequence for GFP-dgn and GFP-dgnFS by PCR according to the protocol of the pENTR Directional TOPO Cloning Kit and continue with the pLenti6/V5 Directional TOPO Cloning Kit (Figure 6). Virus Production1. The day before transfection (Day 1) seed out HEK 293FT cells in a T75 flasks so that they will be 90-95% confluent on the day of transfection. 2. On the day of transfection dilute 36 μl of lipofectamine 2000 Reagent in 1.5 ml of DMEM without serum and antibiotics in a 15 ml falcon.Use another 15 ml falcon and dilute 3 μg pLenti6/V5 carrying the complementary DNA construct for either GFP-dgn or GFP-dgnFS, 2.5 μg envelope encoding plasmid pMD2.G and 7.5 μg vector backbone psPAX2 in 1.5 ml of DMEM without serum and antibiotics. After 5 min the diluted DNA is combined with the diluted lipofectamine 2000 Reagent. 3. Incubate the mixture for 20 min at room temperature to allow the DNA-lipofectamine complexes to form. 4. Remove the medium of HEK 293FT cells, replace it carefully by 7 ml of medium (without antibiotics) and add the DNA-lipofectamine mixture to the flask and rock it back and forth for mixing (Day 2). Incubate t...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Greussing¹,

Unterluggauer²,

Kozieł³

et al. 2012

JoVE

Self Cite

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“…The role of deubiquitinases in aging of higher organisms remains unknown. In senescent cells, the levels of many ubiquitylated and sumoylated proteins are upregulated including telomerase, p53, histone H2A and PCNA resulting in proteasomal degradation (Grillari et al, 2010). Ubiquitylation of DAF-16 (discussed later), an important longevity protein causes shorter lifespan in worms (Li et al, 2007).…”

Section: Evidence For Epigenetic Changes In Agingmentioning

confidence: 99%

Epigenetic Mechanisms of Longevity and Aging

Sen

Shah

Nativio

et al. 2016

Cell

731

591

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Summary Aging is an inevitable outcome of life, characterized by progressive decline in tissue and organ function and increased risk of mortality. Accumulating evidence links aging to genetic and epigenetic alterations. Given the reversible nature of epigenetic mechanisms, these pathways provide promising avenues for therapeutics against age-related decline and disease. In this review, we provide a comprehensive overview of epigenetic studies from invertebrate organisms, vertebrate models, tissue and in vitro systems. We establish links between common operative aging pathways and hallmark chromatin signatures that can be used to identify “druggable” targets to counter human aging and age-related disease.

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“…Conversely, overexpression of SNEV provokes extension of the replicative lifespan in these cells. In addition to being involved in spliceosome assembly and stabilization, SNEV regulates proteasome function due to its U3-ligase activity, and is involved in DNA repair (Grillari et al, 2010). Furthermore, the haploinsufficiency of SNEV has been demonstrated using a heterozygous mouse model, which showed that the proliferative and repopulating capacity of hematopoietic stem cells from these mice was clearly compromised (Schraml et al, 2008).…”

Section: Splicing Factors Involved In Endothelial Senescencementioning

confidence: 99%

The Splicing Factor SRSF1 as a Marker for Endothelial Senescence

Blanco¹,

Bernabéu²

2012

Front. Physio.

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Aging is the major risk factor per se for the development of cardiovascular diseases. The senescence of the endothelial cells (ECs) that line the lumen of blood vessels is the cellular basis for these age-dependent vascular pathologies, including atherosclerosis and hypertension. During their lifespan, ECs may reach a stage of senescence by two different pathways; a replicative one derived from their preprogrammed finite number of cell divisions; and one induced by stress stimuli. Also, certain physiological stimuli, such as transforming growth factor-β, are able to modulate cellular senescence. Currently, the cellular aging process is being widely studied to identify novel molecular markers whose changes correlate with senescence. This review focuses on the regulation of alternative splicing mediated by the serine–arginine splicing factor 1 (SRSF1, or ASF/SF2) during endothelial senescence, a process that is associated with a differential subcellular localization of SRSF1, which typically exhibits a scattered distribution throughout the cytoplasm. Based on its senescence-dependent involvement in alternative splicing, we postulate that SRSF1 is a key marker of EC senescence, regulating the expression of alternative isoforms of target genes such as endoglin (ENG), vascular endothelial growth factor A (VEGFA), tissue factor (T3), or lamin A (LMNA) that integrate in a common molecular senescence program.

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Post-Translational Modification of Cellular Proteins by Ubiquitin and Ubiquitin-Like Molecules: Role in Cellular Senescence and Aging

Cited by 31 publications

References 246 publications

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Epigenetic Mechanisms of Longevity and Aging

The Splicing Factor SRSF1 as a Marker for Endothelial Senescence

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