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Neuronal apoptosis induced by survival factor deprivation is strongly regulated at the transcriptional level. Notably, the nuclear factor of activated T cell (NFAT) transcription factors have an important role in the control of the survival/death fate of neurons. However, the mechanisms that regulate NFAT activity in response to apoptotic stimuli and the target genes that mediate their effect on neuronal apoptosis are mostly unknown. In a previous study, we identified Trim17 as a crucial E3 ubiquitin ligase that is necessary and sufficient for neuronal apoptosis. Here, we show that Trim17 binds preferentially SUMOylated forms of NFATc3. Nonetheless, Trim17 does not promote the ubiquitination/degradation of NFATc3. NFAT transcription factors are regulated by calcium/calcineurin-dependent nuclear-cytoplasmic shuttling. Interestingly, Trim17 reduced by twofold the calcium-mediated nuclear localization of NFATc3 and, consistent with this, halved NFATc3 activity, as estimated by luciferase assays and by measurement of target gene expression. Trim17 also inhibited NFATc4 nuclear translocation and activity. NFATc4 is known to induce the expression of survival factors and, as expected, overexpression of NFATc4 protected cerebellar granule neurons from serum/KCl deprivation-induced apoptosis. Inhibition of NFATc4 by Trim17 may thus partially mediate the proapoptotic effect of Trim17. In contrast, overexpression of NFATc3 aggravated neuronal death, whereas knockdown of NFATc3 protected neurons from apoptosis. This proapoptotic effect of NFATc3 might be due to a feedback loop in which NFATc3, but not NFATc4, induces the transcription of the proapoptotic gene Trim17. Indeed, we found that overexpression or silencing of NFATc3, respectively, increased or decreased Trim17 levels, whereas NFATc4 had no significant effect on Trim17 expression. Moreover, we showed that NFATc3 binds to the promoter of the Trim17 gene together with c-Jun. Therefore, our results describe a novel mechanism regulating NFAT transcription factors beyond the calcium/calcineurin-dependent pathway and provide a possible explanation for the opposite effects of NFATc3 and NFATc4 on neuronal apoptosis. [ABSTRACT FROM AUTHOR]

Short-term proteasome inhibition has been shown to prevent neuronal apoptosis. However, the key pro-survival proteins that must be degraded for triggering neuronal death are mostly unknown. Here, we show that Mcl-1, an anti-apoptotic Bcl-2 family member, is degraded by the proteasome during neuronal apoptosis. Using primary cultures of cerebellar granule neurons deprived of serum and KCl, we found that ubiquitination and proteasomal degradation of Mcl-1 depended on its prior phosphorylation by GSK3, providing the first insight into post-translational regulation of Mcl-1 in neurons. In a previous study, we have reported that the E3 ubiquitin-ligase Trim17 is both necessary and sufficient for neuronal apoptosis. Here, we identified Trim17 as a novel E3 ubiquitin-ligase for Mcl-1. Indeed, Trim17 co-immunoprecipitated with Mcl-1. Trim17 ubiquitinated Mcl-1 in vitro. Overexpression of Trim17 decreased the protein level of Mcl-1 in a phosphorylation- and proteasome-dependent manner. Finally, knock down of Trim17 expression reduced both ubiquitination and degradation of Mcl-1 in neurons. Moreover, impairment of Mcl-1 phosphorylation, by kinase inhibition or point mutations, not only decreased ubiquitination and degradation of Mcl-1, but also blocked the physical interaction between Trim17 and Mcl-1. As this stabilization of Mcl-1 increased its neuroprotective effect, our data strongly suggest that Trim17-mediated ubiquitination and degradation of Mcl-1 is necessary for initiating neuronal death. [ABSTRACT FROM AUTHOR]

Neuronal apoptosis has a major role during development and aberrant apoptosis contributes to the pathology of certain neurological conditions. Studies with nerve growth factor (NGF)-dependent sympathetic neurons have provided important insights into the molecular mechanisms of neuronal apoptosis and the signalling pathways that regulate the cell death programme in neurons. The BH3-only protein Bim is a critical mediator of apoptosis in many cell types and in sympathetic neurons is required for NGF withdrawal-induced death. However, regulation of bim expression is complex and remains incompletely understood. We report that a conserved inverted CCAAT box (ICB) in the rat bim promoter is bound by the heterotrimeric transcription factor NF-Y. Interestingly, NF-Y is required for bim promoter activity and its induction following NGF withdrawal. We demonstrate that NF-Y activity is essential for endogenous Bim expression and contributes to NGF withdrawal-induced death. Furthermore, we find that the transcriptional coactivators CBP and p300 interact with NF-Y and FOXO3a and bind to this region of the bim promoter. The amount of CBP/p300 bound to bim increases after NGF deprivation and inhibition of CBP/p300 activity reduces bim induction. Our results indicate that NF-Y cooperates with FOXO3a to recruit CBP/p300 to the bim promoter to form a stable multi-protein/DNA complex that activates bim transcription after survival factor withdrawal. [ABSTRACT FROM AUTHOR]

Abstract Background Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. Results Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. Conclusion These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.

NFATc3 is the predominant member of the NFAT family of transcription factors in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 binds and ubiquitinates NFATc3 in vitro and in cells where it reduces NFATc3 protein level and transcriptional activity. In contrast, silencing of endogenous Trim39 decreases NFATc3 ubiquitination and increases its activity, thereby resulting in enhanced neuronal apoptosis. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, we identify Trim39 as a new SUMO-targeted E3 ubiquitin-ligase (STUbL). Indeed, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motifs in Trim39 reduces NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. In addition, Trim39 preferentially ubiquitinates SUMOylated forms of NFATc3 in vitro. As a consequence, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity in neurons. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a STUbL for NFATc3., (© 2022. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

TRIM17 is a member of the TRIM family, a large class of RING-containing E3 ubiquitin-ligases. It is expressed at low levels in adult tissues, except in testis and in some brain regions. However, it can be highly induced in stress conditions which makes it a putative stress sensor required for the triggering of key cellular responses. As most TRIM members, TRIM17 can act as an E3 ubiquitin-ligase and promote the degradation by the proteasome of substrates such as the antiapoptotic protein MCL1. Intriguingly, TRIM17 can also prevent the ubiquitination of other proteins and stabilize them, by binding to other TRIM proteins and inhibiting their E3 ubiquitin-ligase activity. This duality of action confers several pivotal roles to TRIM17 in crucial cellular processes such as apoptosis, autophagy or cell division, but also in pathological conditions as diverse as Parkinson's disease or cancer. Here, in addition to recent data that endorse this duality, we review what is currently known from public databases and the literature about TRIM17 gene regulation and expression, TRIM17 protein structure and interactions, as well as its involvement in cell physiology and human disorders.

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.

Although accumulating data indicate that increased α-synuclein expression is crucial for Parkinson disease (PD), mechanisms regulating the transcription of its gene, SNCA, are largely unknown. Here, we describe a pathway regulating α-synuclein expression. Our data show that ZSCAN21 stimulates SNCA transcription in neuronal cells and that TRIM41 is an E3 ubiquitin ligase for ZSCAN21. In contrast, TRIM17 decreases the TRIM41-mediated degradation of ZSCAN21. Silencing of ZSCAN21 and TRIM17 consistently reduces SNCA expression, whereas TRIM41 knockdown increases it. The mRNA levels of TRIM17, ZSCAN21, and SNCA are simultaneously increased in the midbrains of mice following MPTP treatment. In addition, rare genetic variants in ZSCAN21, TRIM17, and TRIM41 genes occur in patients with familial forms of PD. Expression of variants in ZSCAN21 and TRIM41 genes results in the stabilization of the ZSCAN21 protein. Our data thus suggest that deregulation of the TRIM17/TRIM41/ZSCAN21 pathway may be involved in the pathogenesis of PD., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Glycogenin-interacting protein 1 (GNIP1) is a tripartite motif (TRIM) protein with E3 ubiquitin ligase activity that interacts with glycogenin. These data suggest that GNIP1 could play a major role in the control of glycogen metabolism. However, direct evidence based on functional analysis remains to be obtained., Objectives: The aim of this study was 1) to define the expression pattern of glycogenin-interacting protein/Tripartite motif containing protein 7 (GNIP/TRIM7) isoforms in humans, 2) to test their ubiquitin E3 ligase activity, and 3) to analyze the functional effects of GNIP1 on muscle glucose/glycogen metabolism both in human cultured cells and in vivo in mice., Results: We show that GNIP1 was the most abundant GNIP/TRIM7 isoform in human skeletal muscle, whereas in cardiac muscle only TRIM7 was expressed. GNIP1 and TRIM7 had autoubiquitination activity in vitro and were localized in the Golgi apparatus and cytosol respectively in LHCN-M2 myoblasts. GNIP1 overexpression increased glucose uptake in LHCN-M2 myotubes. Overexpression of GNIP1 in mouse muscle in vivo increased glycogen content, glycogen synthase (GS) activity and phospho-GSK-3α/β (Ser21/9) and phospho-Akt (Ser473) content, whereas decreased GS phosphorylation in Ser640. These modifications led to decreased blood glucose levels, lactate levels and body weight, without changing whole-body insulin or glucose tolerance in mouse., Conclusion: GNIP1 is an ubiquitin ligase with a markedly glycogenic effect in skeletal muscle., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17) and one deubiquitinase (e.g., USP9X), that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter.

The Ras-association domain family 1 (RASSF1) gene has seven different isoforms; isoform A is a tumor-suppressor gene (RASSF1A). The promoter of RASSF1A is inactivated in many cancers, whereas the expression of another major isoform, RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A, interacts with betaTrCP. Binding of RASSF1C to betaTrCP involves serine 18 and serine 19 of the SS(18)GYXS(19) motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by betaTrCP; however, surprisingly, the association between RASSF1C and betaTrCP does not occur via the betaTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the beta-catenin oncogene, due to inhibition of its betaTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for beta-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on beta-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis.

ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF(betaTrCP) ubiquitin ligase that binds to betaTrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.

The human immunodeficiency virus type 1 Vpu protein acts as an adaptor for the proteasomal degradation of CD4 by recruiting CD4 and beta-transducin repeat-containing protein (betaTrCP), the receptor component of the multisubunit SCF-betaTrCP E3 ubiquitin ligase complex. We showed that the expression of a Vpu-green fluorescent fusion protein prevented the proteosomal degradation of betaTrCP substrates such as beta-catenin, IkappaBalpha, and ATF4, which are normally directly targeted to the proteasome for degradation. Beta-catenin was translocated into the nucleus, whereas the tumor necrosis factor-induced nuclear translocation of NFkappaB was impaired. Beta-catenin was also up-regulated in cells producing Vpu+ human immunodeficiency virus type 1 but not in cells producing Vpu-deficient viruses. The overexpression of ATF4 also provoked accumulation of beta-catenin, but to a lower level than that resulting from the expression of Vpu. Finally, the expression of Vpu induces the exclusion of betaTrCP from the nucleus. These data suggest that Vpu is a strong competitive inhibitor of betaTrCP that impairs the degradation of SCFbetaTrCP substrates as long as Vpu has an intact phosphorylation motif and can bind to betaTrCP.

The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein betaTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IkappaBalpha and beta-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of betaTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of betaTrCP. The F-box-deleted betaTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCF(betaTrCP) complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.