Cul-4 inhibition rescues spastin levels and reduces defects in hereditary spastic paraplegia models.

Hereditary spastic paraplegias (HSPs) are degenerative motor neuron diseases characterized by progressive spasticity and weakness in the lower limbs. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme spastin. Although, there is no cure for SPG4-HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Spastin protein levels are regulated by poly-ubiquitination and proteasomal-mediated degradation, in a neddylation-dependent manner. However, the molecular players involved in this regulation are unknown. Here, we show that the Cullin-4-Ring E3 ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation in spastin-proficient and in patient derived SPG4 haploinsufficient cells. To evaluate the role of CRL4 complex in spastin regulation in vivo, we developed a Drosophila melanogaster model of SPG4 haploinsufficiency which show alterations of synapse morphology and locomotor activity, recapitulating phenotypical defects observed in patients. Downregulation of the CRL4 complex, highly conserved in Drosophila, rescues spastin levels and the phenotypical defects observed in flies. As a proof of concept of possible pharmacological treatments, we demonstrate a recovery of spastin levels and amelioration of the SPG4-HSP-associated defects both in the fly model and in patient-derived cells by chemical inactivation of the CRL4 complex with NSC1892. Taken together, these findings show that CRL4 contributes to spastin stability regulation and that it is possible to induce spastin recovery and rescue of SPG4-HSP defects by blocking the CRL4-mediated spastin degradation.

Neuro-Immune Modulation of Cholinergic Signaling in an Addiction Vulnerability Trait.

Sign-tracking (ST) describes the propensity to approach and contact a Pavlovian reward cue. By contrast, goal-trackers (GTs) respond to such a cue by retrieving the reward. These behaviors index the presence of opponent cognitive-motivational traits, with STs exhibiting attentional control deficits, behavior dominated by incentive motivational processes, and vulnerability for addictive drug taking. Attentional control deficits in STs were previously attributed to attenuated cholinergic signaling, resulting from deficient translocation of intracellular choline transporters (CHTs) into synaptosomal plasma membrane. Here, we investigated a posttranslational modification of CHTs, poly-ubiquitination, and tested the hypothesis that elevated cytokine signaling in STs contributes to CHT modification. We demonstrated that intracellular CHTs, but not plasma membrane CHTs, are highly ubiquitinated in male and female sign-tracking rats when compared with GTs. Moreover, levels of cytokines measured in cortex and striatum, but not spleen, were higher in STs than in GTs. Activation of the innate immune system by systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) elevated ubiquitinated CHT levels in cortex and striatum of GTs only, suggesting ceiling effects in STs. In spleen, LPS increased levels of most cytokines in both phenotypes. In cortex, LPS particularly robustly increased levels of the chemokines CCL2 and CXCL10. Phenotype-specific increases were restricted to GTs, again suggesting ceiling effects in STs. These results indicate that interactions between elevated brain immune modulator signaling and CHT regulation are essential components of the neuronal underpinnings of the addiction vulnerability trait indexed by sign-tracking.

TRIM8 inhibits breast cancer proliferation by regulating estrogen signaling.

Breast cancer (BC) is the most common female malignancy worldwide, and 70% of which are estrogen receptor α (ERα) positive. Endocrine treatment, such as tamoxifen, is a primary adjuvant therapy for patients with ER-positive BC. However, some patients will develop acquired resistance following long-time treatment. Further research on estrogen signaling is important to improve the therapy of these patients. In this study, we report that the E3 ubiquitin ligase tripartite motif 8 (TRIM8) acts as a novel regulator of ERα signaling. TRIM8 is downregulated in BC and is associated with poor prognosis. In addition, the protein level of TRIM8 is negatively correlated with ERα. RNA sequencing revealed that estrogen signaling maybe a potential target of TRIM8. Moreover, knockdown of TRIM8 can significantly enhance BC cell proliferation and migration both in vitro and in vivo. And this effect can be reversed by ERα depletion. Further mechanistic studies showed that TRIM8 interacts with AF1 domain of ERα via its RING domain in the cytoplasm and increases poly-ubiquitination of the ERα protein. In conclusion, our study reveals an interesting post-translational mechanism between ERα and TRIM8 in ER-positive BC, which suggests that TRIM8 may be a potential therapeutic target in the treatment of BC.

Uev1A amino terminus stimulates poly-ubiquitin chain assembly and is required for NF-κB activation.

The ubiquitin (Ub)-conjugating enzyme variants (Uev) Uev1A and Mms2 interact with Ubc13 to form heterodimeric complexes with different biological functions. Uev1A-Ubc13 is involved in NF-κB activation while Mms2-Ubc13 is required for the DNA-damage response. The structural comparison of the core domains of these two Uevs reveals no obvious difference, suggesting that the amino terminal extension of Uev1A plays a critical role in the functional determination. Indeed, truncated Uev1A lacking the N-terminal extension behaves like Mms2, while a chimeric protein containing the N-terminal Uev1A fused to Mms2 functionally resembles Uev1A. Interestingly, the N-terminal extension of Uev1A also dictates whether to assemble di- or poly-Ub chains in an in vitro reaction. Both thermodynamic measurements and enzymatic assays revealed that the Uev1A N-terminal extension weakens the Uev-Ubc13 interaction; however, other means capable of causing a reduced Uev1A-Ubc13 affinity and poly-Ub chain assembly do not necessarily promote NF-κB activation, indicating that the poly-Ub chain formation is not the only component contributed by the N-terminal extension of Uev1A. The physiological relevance of the Uev1A N-terminal truncation is presented and discussed.

The Lysosome Pathway Degrades CD81 on the Cell Surface by Poly-ubiquitination and Clathrin-Mediated Endocytosis.

CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway via endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD81 half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMP1 and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD81 expression on the cell surface. Hence, we demonstrated that CD81 is internalized by clathrin-mediated endocytosis and subsequently degraded via a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.

HIF-1α Upregulation due to Depletion of the Free Ubiquitin Pool.

Hypoxia-inducible factor 1alpha (HIF-1α), which transactivates a variety of hypoxia-induced genes, is rapidly degraded under nomoxia through the hydroxylation-ubiquitination-proteasome pathway. In this study, we addressed how HIF-1α is stabilized by proteasome inhibitors. The ubiquitin pool was rapidly reduced after proteasome inhibition, followed by the accumulation of non-ubiquitinated HIF-1α. The poly-ubiquitination of HIF-1α was resumed by restoration of free ubiquitin, which suggests that the HIF-1α stabilization under proteasome inhibition is attributed to depletion of the free ubiquitin pool. Ni(2+) and Zn(2+) also stabilized HIF-1α with depletion of the free ubiquitin pool and these effects of metal ions were attenuated by restoration of free ubiquitin. Ni(2+) and Zn(2+) may disturb the recycling of free ubiquitin, as MG132 does. Based on these results, the state of the ubiquitin pool seems to be another critical factor determining the cellular level of HIF-1α.

HIF-1alpha Upregulation due to Depletion of the Free Ubiquitin Pool

Hypoxia-inducible factor 1alpha (HIF-1alpha), which transactivates a variety of hypoxia-induced genes, is rapidly degraded under nomoxia through the hydroxylation-ubiquitination-proteasome pathway. In this study, we addressed how HIF-1alpha is stabilized by proteasome inhibitors. The ubiquitin pool was rapidly reduced after proteasome inhibition, followed by the accumulation of non-ubiquitinated HIF-1alpha. The poly-ubiquitination of HIF-1alpha was resumed by restoration of free ubiquitin, which suggests that the HIF-1alpha stabilization under proteasome inhibition is attributed to depletion of the free ubiquitin pool. Ni2+ and Zn2+ also stabilized HIF-1alpha with depletion of the free ubiquitin pool and these effects of metal ions were attenuated by restoration of free ubiquitin. Ni2+ and Zn2+ may disturb the recycling of free ubiquitin, as MG132 does. Based on these results, the state of the ubiquitin pool seems to be another critical factor determining the cellular level of HIF-1alpha.