Integrated Regulation of HuR by Translation Repression and Protein Degradation Determines Pulsatile Expression of p53 Under DNA Damage

Guha, Abhishek; Ahuja, Deepika; Mandal, Sukhen Das; Parasar, Bibudha; Deyasi, Krishanu; Roy, Debadrita; Sharma, Vasundhara; Willard, Belinda; Ghosh, Anandamohan; Ray, Partho Sarothi

doi:10.1016/j.isci.2019.05.002

Cited by 36 publications

(45 citation statements)

References 52 publications

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“…It is curious that HO2 protein degradation levels plateau at around 50%, a phenomenon that is not unique to HO2 (25,26). We propose that, since HO2 possesses critical cytoprotective functions, an unknown factor(s) is induced to protect it from further degradation when a threshold low cellular concentration is reached.…”

Section: Resultsmentioning

confidence: 97%

Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

Liu

Dumbrepatil

Fleischhacker

et al. 2020

Journal of Biological Chemistry

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Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site, and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2’s role in maintaining heme homeostasis, paving the way for future investigation into HO2’s pathophysiological role in heme deficiency response.

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Section: Resultsmentioning

confidence: 97%

Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

Liu

Dumbrepatil

Fleischhacker

et al. 2020

Journal of Biological Chemistry

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“…As TRIM21 has been shown to interact with HuR and mediates ubiquitination-dependent proteasomal degradation of HuR under genotoxic stress 26 , we sought to understand in more detail the cellular function of TRIM21 in the context of DNA damage. We found that TRIM21 knockdown abrogates the decrease of HuR post 4 h of UV exposure (Fig.…”

Section: Trim21 Reverses the Decrease In Cell Proliferation And Colonmentioning

confidence: 99%

“…TRIM21 also promotes the proliferation of osteosarcoma cells and its expression enhances the tolerance of osteosarcoma cells to various stresses 25 . It also affects the expression and stability of p53 in different cancers [26][27][28] . Recently, TRIM21 has been shown to cause the temporally-regulated degradation of the RNA-binding protein HuR in response to UVC irradiation.…”

mentioning

confidence: 99%

“…Recently, TRIM21 has been shown to cause the temporally-regulated degradation of the RNA-binding protein HuR in response to UVC irradiation. The negative regulation of HuR by TRIM21 contributes to the pulsatile change in p53 protein level in response to UV irradiation, thereby limiting the expression of p53 under DNA damage 26 . However, the physiological role of TRIM21 in response to DNA damage has not been elucidated.…”

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confidence: 99%

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Negative feedback regulation by HuR controls TRIM21 expression and function in response to UV radiation

Guha

Nag

Ray

2020

Sci Rep

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The E3 ubiquitin ligase TRIM21 plays a crucial role as a negative regulator of innate immune responses. Recent evidence has also indicated the involvement of TRIM21 in the genotoxic stress response and suppressing tumorigenesis. Our previous work has demonstrated a new function of TRIM21 in inhibiting p53 protein synthesis by degrading the RNA-binding protein HuR in response to UV radiation. This suggested a pro-oncogenic role of TRIM21. In this study, we have shown that TRIM21 enhances the proliferation of MCF7 breast carcinoma cells and counteracts the decrease in cell proliferation and colony formation caused by UV-induced DNA damage. Further, this pro-oncogenic role of TRIM21 in response to DNA damage is mediated by its degradation of HuR. Conversely, we found that HuR binds to a U-rich element in the 3′UTR of TRIM21 mRNA and activates its translation, thereby constituting a negative feedback loop. We found that dihydrotanshinone-I (DHTS-I), a plantderived product which prevents HuR binding to specific RNAs, prevented HuR-mediated upregulation of TRIM21, while increasing the HuR-mediated upregulation of p53. Together, these findings demonstrate a negative feedback regulation between TRIM21 and HuR, which may play an important role in regulating the level of p53 in the genotoxic stress response. TRIM (Tripartite motif) family proteins are ubiquitously found in all metazoans and are recognized for conserved biological functions, such as control of cell proliferation and differentiation. However, with the appearance of complex immune systems in jawed vertebrates, some of these have evolved to directly regulate antiviral action and cytokine production. TRIM family proteins are primarily recognized as post-translational modifiers. Nevertheless, some of the members of this family are implicated in RNA metabolism. These TRIM proteins are specifically localized in the cytoplasmic processing bodies. Furthermore, about half of all the TRIM family proteins are involved in autophagy. Most of these are also involved in the formation of TRIMosomes which are considered to facilitate autophagic engulfment and subsequent lysosomal degradation 1. E3 ubiquitin ligase TRIM21 is a 52 kDa protein belonging to the TRIM family. Similar to many other TRIM family proteins, TRIM21 has a characteristic N-terminal RING domain, B-box domain, coiled-coil domain (collectively called RBCC). The RING domain is implicated in E3 ubiquitin ligase activity, whereas the B-box domain has been shown to be a negative regulator of RING domain. Recently, the phosphorylation of serine-80 (S80) or phosphomimetic serine-80 to glutamate (S80E) mutation in TRIM21 has been found to release the inhibition of B-box domain 2,3. The coiled-coil domain is utilized in homo-dimerization of TRIM21 which ensures binding with the substrate in a stoichiometry of 2:1 2. TRIM21 has also C-terminal PRYSPRY domain which is required for target protein recognition. Structural analysis of PRYSPRY domain of TRIM21 has demonstrated its diverse functions. Isothermal titration ca...

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“…Interaction of Hur and Trim21 (Guha et al, 2019) were used as used as positive controls and 249 secondary antibodies against ATP7B (anti-rabbit) and VPS35 (anti-goat) were used as negative 250 controls. We observed positive interaction between ATP7B and VPS35 that is evident by 251 formation of red intracellular puncta on sites if the two proteins juxtapose at a distance less than 252 <40nm ( Fig 7F).…”

mentioning

confidence: 99%

Retromer retrieves the Wilson Disease protein, ATP7B from lysosomes in a copper-dependent mode

Gupta

Das

Maji

et al. 2020

Preprint

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localizes with lysosomal marker, Lamp1 and the core member of retromer complex, Vps35. 11Knocking down VPS35 did not alter copper-responsive vesicularization of ATP7B; rather upon 12 subsequent copper chelation, ATP7B failed to relocalize to TGN that could be rescued by 13 overexpressing wtVPS35. Using super-resolution microscopy and proximity ligation assays we 14 demonstrate that VPS35 and ATP7B are juxtaposed on the same lysosomal compartment and 15 their interaction is indirect. Utilizing in-cell photoamino acid-based UV-crosslinking and 16 subsequent immunoprecipitation, we detected ATP7B and retromer subunits, VPS35 and 17 VPS26 in a large complex in high copper conditions, hence confirming their interaction. We 18 demonstrate that retromer regulates lysosome to TGN trafficking of the copper transporter 19 ATP7B and it is dependent upon cellular copper level. 20

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Integrated Regulation of HuR by Translation Repression and Protein Degradation Determines Pulsatile Expression of p53 Under DNA Damage

Cited by 36 publications

References 52 publications

Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

Negative feedback regulation by HuR controls TRIM21 expression and function in response to UV radiation

Retromer retrieves the Wilson Disease protein, ATP7B from lysosomes in a copper-dependent mode

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