Analysis of Protein Stability by the Cycloheximide Chase Assay

Kao, Shih-Han; Wang, Wen-Lung; Chen, Chi-Yuan; Chang, Yih‐Leong; Wu, Yi-Ying; Wang, Yiting; Wang, Shuping; Nesvizhskii, Alexey I.; Chen, Yu‐Ju; Hong, Tse-Ming; Yang, Pan-Chyr

doi:10.21769/bioprotoc.1374

Cited by 97 publications

(66 citation statements)

References 2 publications

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“…31 Proteasomal blockade with either epoximicin or MG-132 led to a significant increase in the intracellular halflife of BMP2 following epoximicin treatment (Figure 2A; **P ≤ 0.01 at 30-and 60-min posttreatment with epoximicin and *P ≤ 0.05 at 30 and 60 minutes post-MG-132). 293T cells were transfected with a plasmid vector, expressing BMP2 and treated with cycloheximide (Chx) in presence or absence of proteasomal inhibitors.…”

Section: Half-life Through Posttranslational Mechanismsmentioning

confidence: 91%

“…Cycloheximide blocks de novo synthesis of proteins, allowing measurement of their steady-state turnover. 31 Proteasomal blockade with either epoximicin or MG-132 led to a significant increase in the intracellular halflife of BMP2 following epoximicin treatment (Figure 2A; **P ≤ 0.01 at 30-and 60-min posttreatment with epoximicin and *P ≤ 0.05 at 30 and 60 minutes post-MG-132). Addition of epoximicin and MG-132 led to significant delay in BMP2 degradation, almost doubling BMP2 half-life (Figure 2A; 30min post-cycloheximide addition in comparison to >60 minutes following addition of epoximicin or MG-132, *P ≤ 0.05 to P ≤ 0.01).…”

Section: Half-life Through Posttranslational Mechanismsmentioning

confidence: 91%

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Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion

et al. 2019

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The short half-life and use of recombinant bone morphogenetic protein (BMP)2 in large doses poses major limitations in the clinic. Events regulating posttranslational processing and degradation of BMP2 in situ, linked to its secretion, have not been understood. Toward identifying mechanisms regulating intracellular BMP2 stability, we first discovered that inhibiting proteasomal degradation enhances both intracellular BMP2 level and its extracellular secretion. Next, we identified BMP2 degradation occurs through an ubiquitin-mediated mechanism. Since ubiquitination precedes proteasomal turnover and mainly occurs on lysine residues of nascent proteins, we systematically mutated individual lysine residues within BMP2 and tested them for enhanced stability. Results revealed that substitutions on four lysine residues within the pro-BMP2 region and three in the mature region increased both BMP2 stability and extracellular secretion. Structural modeling revealed key lysine residues involved in proteasomal degradation occupy a lysine cluster near proprotein convertase cleavage site. Interestingly, mutations within these residues did not affect biological activity of BMP2. These data suggest that preventing intracellular proteasomal loss of BMP2 through genetic modifications can overcome limitations related to its short half-life. K E Y W O R D SBMP2, proteasomal degradation, protein turnover, ubiquitination | 181 KHATTAR eT Al.

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Section: Half-life Through Posttranslational Mechanismsmentioning

confidence: 91%

Section: Half-life Through Posttranslational Mechanismsmentioning

confidence: 91%

Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion

et al. 2019

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“…The CHX chase experiment was performed as described previously (60). Cells were seeded on 35-mm culture dishes at a density of 6 ϫ 10 6 cells.…”

Section: Cellmentioning

confidence: 99%

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

Liu

Zheng

Shu

et al. 2016

J Virol

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Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro. Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCEInfection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro. Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication. S mall ubiquitin-like modifier (SUMO) modification, a member of the group of posttranslational modifications (PTMs), is important for the regulation of many cellular proteins and pathways (1). SUMO is a novel...

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“…PP2CA plants ( Kao et al, 2015). As shown in Figure 1(b), PP2CA proteins were more degraded with ABA treatment versus no treatment.…”

Section: Resultsmentioning

confidence: 89%

“…To determine whether the PP2CA is degraded via the ubiquitin‐26S proteasome pathway, we treated each reaction mixture with the proteasome inhibitor MG132 (Joo et al ., ) and found that MG132 inhibited the degradation of the PP2CA protein. Moreover, we investigated the stability of PP2CA in vivo in the absence of de novo protein synthesis through cycloheximide (CHX) chase assay using 35S:: PP2CA plants (Kao et al ., ). As shown in Figure (b), PP2CA proteins were more degraded with ABA treatment versus no treatment.…”

Section: Resultsmentioning

confidence: 99%

The RING finger E3 ligases PIR1 and PIR2 mediate PP2CA degradation to enhance abscisic acid response in Arabidopsis

et al. 2019

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Summary Recent work has established a core ABA signaling pathway in which A‐type PP2C protein phosphatases act as central negative modulators. Although ABA signaling inhibits PP2C activity through ABA‐receptor complex, it remains unknown if other mechanisms exist to modulate the level of PP2Cs. Here, we identified a RING domain ubiquitin E3 ligase, PIR1 (PP2CA interacting RING finger protein 1), that interacted with PP2CA. Of the two splicing isoforms, PIR1.2 was isolated from leaf tissue. The PIR1.2 exhibited E3 ligase activity and determined PP2CA stability in the presence of ABA. Consistent with the conclusion that PIR1 promotes ABA signaling by removing PP2CA, a negative modulator, the pir1 knockout mutant displayed an ABA‐hyposensitive phenotype. We further showed that PIR2, the closest homologue of PIR1.2, also interacted with PP2CA. Although the pir2 knockout mutant did not display altered ABA response, the pir1‐1/pir2 double mutant became more insensitive to ABA than the wild‐type or pir1‐1 and pir2 single mutants. Using a cell‐free degradation assay, ABA promoted degradation of PP2CA, however, such degradation was delayed when incubated with protein extract prepared from the pir1‐1/pir2 double mutant. Our data suggest that PIR1 and PIR2 positively modulate ABA signaling by targeting PP2CA for degradation.

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Analysis of Protein Stability by the Cycloheximide Chase Assay

Cited by 97 publications

References 2 publications

Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion

Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

The RING finger E3 ligases PIR1 and PIR2 mediate PP2CA degradation to enhance abscisic acid response in Arabidopsis

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