TDP-43 Cytoplasmic Translocation in the Skin Fibroblasts of ALS Patients

Diagnosis of ALS is based on clinical symptoms when motoneuron degeneration is significant. Therefore, new approaches for early diagnosis are needed. We aimed to assess if alterations in appearance and cellular localization of cutaneous TDP-43 may represent a biomarker for ALS. Skin biopsies from 64 subjects were analyzed: 44 ALS patients, 10 healthy controls (HC) and 10 neurological controls (NC) (Parkinson’s disease and multiple sclerosis). TDP-43 immunoreactivity in epidermis and dermis was analyzed, as well as the percentage of cells with TDP-43 cytoplasmic localization. We detected a higher amount of TDP-43 in epidermis (p < 0.001) and in both layers of dermis (p < 0.001), as well as a higher percentage of TDP-43 cytoplasmic positive cells (p < 0.001) in the ALS group compared to HC and NC groups. Dermal cells containing TDP-43 were fibroblasts as identified by co-labeling against vimentin. ROC analyses (AUC 0.867, p < 0.001; CI 95% 0.800–0.935) showed that detection of 24.1% cells with cytoplasmic TDP-43 positivity in the dermis had 85% sensitivity and 80% specificity for detecting ALS. We have identified significantly increased TDP-43 levels in epidermis and in the cytoplasm of dermal cells of ALS patients. Our findings provide support for the use of TDP-43 in skin biopsies as a potential biomarker.

The ADAR1 editome reveals drivers of editing-specificity for ADAR1-isoforms

Adenosine deaminase acting on RNA (ADAR) (also known as ADAR1) promotes A-to-I conversion in double-stranded and highly structured RNAs. ADAR1 has two isoforms transcribed from different promoters: ADAR1p150, which is mainly cytoplasmic and interferon-inducible, and constitutively expressed ADAR1p110 that is primarily localized in the nucleus. Mutations in ADAR1 cause Aicardi – Goutières syndrome (AGS), a severe autoinflammatory disease in humans associated with aberrant IFN production. In mice, deletion of ADAR1 or selective knockout of the p150 isoform alone leads to embryonic lethality driven by overexpression of interferon-stimulated genes. This phenotype can be rescued by concurrent deletion of cytoplasmic dsRNA-sensor MDA5. These findings indicate that the interferon-inducible p150 isoform is indispensable and cannot be rescued by the ADAR1p110 isoform. Nevertheless, editing sites uniquely targeted by ADAR1p150 but also mechanisms of isoform-specificity remain elusive. Here we combine RIP-seq on human cells expressing ADAR1 isoforms and combine this with analysis of isoform-specific editing patterns in genetically modified mouse cells to extensively investigate ADAR1-isoform binding- and editing characteristics. Moreover, using mutated ADAR variants, we examine the effect of two unique features of ADAR1p150 on its target specificity: 1) cytoplasmic localization and 2) Z-DNA binding domain α. Our findings indicate that ZBDa contributes only minimally to p150 editing-specificity and that isoform-specific editing is directed mainly by the cytoplasmic localization of the editase.

Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells

We have identified triptolide as a novel NRF2 inhibitor, which significantly attenuates ARE-luciferase activity at nanomolar concentrations. Triptolide did not affect the level of NRF2, but significantly inhibited the expression of NRF2 target genes in A549 cells. We found that NRF2 possesses a previously unrecognized NES in the Neh2 domain, and that triptolide promotes an interaction between NRF2 and CRM1. Triptolide also decreased nuclear accumulation of NRF2, suggesting that it promotes nuclear export of NRF2. In addition, we show that triptolide decreased the expression of NRF2 target genes and increased intracellular oxidative stress, suppressing invasion and promoting cisplatin-induced apoptosis in A549 cells. Finally, oral administration of triptolide suppressed the growth of A549 xenografts in athymic mice by decreasing the expression of NRF2 target genes and promoting oxidative damages via the nuclear export of NRF2 and CRM1 in vivo. To the best of our knowledge, triptolide is the first type of compound to inhibit NRF2 by increasing cytoplasmic localization of NRF2.

Lung eosinophils elicited during allergic and acute aspergillosis express RORγt and IL-23R but do not require IL-23 for IL-17 production

Exposure to the mold, Aspergillus, is ubiquitous and generally has no adverse consequences in immunocompetent persons. However, invasive and allergic aspergillosis can develop in immunocompromised and atopic individuals, respectively. Previously, we demonstrated that mouse lung eosinophils produce IL-17 in response to stimulation by live conidia and antigens of A. fumigatus. Here, we utilized murine models of allergic and acute pulmonary aspergillosis to determine the association of IL-23, IL-23R and RORγt with eosinophil IL-17 expression. Following A. fumigatus stimulation, a population of lung eosinophils expressed RORγt, the master transcription factor for IL-17 regulation. Eosinophil RORγt expression was demonstrated by flow cytometry, confocal microscopy, western blotting and an mCherry reporter mouse. Both nuclear and cytoplasmic localization of RORγt in eosinophils were observed, although the former predominated. A population of lung eosinophils also expressed IL-23R. While expression of IL-23R was positively correlated with expression of RORγt, expression of RORγt and IL-17 was similar when comparing lung eosinophils from A. fumigatus-challenged wild-type and IL-23p19-/- mice. Thus, in allergic and acute models of pulmonary aspergillosis, lung eosinophils express IL-17, RORγt and IL-23R. However, IL-23 is dispensable for production of IL-17 and RORγt.

The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking

Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.

A Missense POU4F3 Variant Associated with Autosomal Dominant Midfrequency Hearing Loss Alters Subnuclear Localization and Transcriptional Capabilities

Background. The pathogenic variant, POU class 4 transcription factor 3 (POU4F3), is reported to cause autosomal dominant nonsyndromic hearing loss (ADNSHL). Previously, we have examined a four-generation midfrequency sensorineural hearing loss (MFSNHL) family (no. 6126) and established POU4F3 c.602T>C (p.Leu201Pro) as a potential disease-causing variant. Objectives. We explored the structural and functional alterations that the c.602T>C (p.Leu201Pro) variant enforces on the POU4F3 protein. Methods. We utilized wild-type (WT) and mutant (MUT) POU4F3 c.602T>C plasmid incorporation into HeLa cells to assess functional changes, by immunofluorescence and luciferase assays. To predict protein structural alterations in the MUT versus WT POU4F3, we also generated 3D structures to compare both types of POU4F3 proteins. Results. The WT POU4F3 is ubiquitously present in the nucleus, whereas the MUT form of POU4F3 exhibits a more restricted nuclear presence. This finding is different from other publications, which report a cytoplasmic localization of the MUT POU4F3. We also demonstrated that, as opposed to WT POU4F3, the MUT POU4F3 had 40% reduced luciferase activity. Conclusions. The reduced nuclear presence, combined with reduced transcriptional activity, suggests that the POU4F3 c.602T>C variant alters cellular activity and may contribute to the pathogenicity of POU4F3-related hearing loss. It, also, provides more evidence of the pathophysiological characteristics of MFSNHL.

Cytoplasmic Localization Isoform of Cyclin Y Enhanced the Metastatic Ability of Lung Cancer via Regulating Tropomyosin 4

Cyclin Y (CCNY) is a novel cyclin and highly conserved in metazoan species. Previous studies from our and other laboratory indicate that CCNY play a crucial role in tumor progression. There are two CCNY isoform which has different subcellular distributions, with cytoplasmic isoform (CCNYc) and membrane distribution isoform (CCNYm). However, the expression and function of CCNY isoforms is still unclear. We firstly found CCNYc was expressed in natural lung cancer tissue and cells through the subcellular distribution. Co-IP and immunofluorescence showed that both CCNYm and CCNYc could interact with PFTK1. Further studies illustrated that CCNYc but not CCNYm enhanced cell migration and invasion activity both in vivo and vitro. The function of CCNYc could be inhibited by suppression of PFTK1 expression. In addition, our data indicated that tropomyosin 4 (TPM4), a kind of actin-binding proteins, was down-regulated by suppression of CCNY. F-actin assembly could be controlled by CCNYc as well as PFTK1 and TPM4. As a result, CCNY was mainly expressed in lung cancer. CCNYc could promote cell motility and invasion. It indicated that CCNYc/PFTK1 complex could promote cell metastasis by regulating the formation of F-actin via TPM4.

The parkinsonism-associated protein FBXO7 cooperates with the BAG6 complex in proteasome function and controls the subcellular localization of the complex

The regulation of proteasome activity is essential to cellular homeostasis and defects have been implicated in various disorders including Parkinson disease. The F-box protein FBXO7 has been implicated in early-onset parkinsonism and has previously been shown to have a regulatory role in proteasome activity and assembly. Here, we report the association of the E3 ubiquitin ligase FBXO7-SCF (SKP1, cullin-1, F-box protein) with the BAG6 complex, consisting of the subunits BAG6, GET4 and UBL4A. We identify the subunit GET4 as a direct interactor of FBXO7 and we show that the subunits GET4 and UBL4A are required for proper proteasome activity. Our findings demonstrate reduced binding of FBXO7 variants to GET4 and that FBXO7 variants bring about reduced proteasome activity. In addition, we find that GET4 is a non-proteolytic substrate of FBXO7, that binding of GET4 to BAG6 is enhanced in the presence of active FBXO7-SCF and that the cytoplasmic localization of the BAG6 complex is dependent on the E3 ubiquitin ligase activity. Taken together, our study shows that the parkinsonism-associated FBXO7 cooperates with the BAG6 complex in proteasome function and determines the subcellular localization of this complex.

Role of cytoplasmic localization of maspin in promoting cell invasion in breast cancer with aggressive phenotype

Mammary serine protease inhibitor (maspin) is a tumor suppressor gene that is downregulated during carcinogenesis and breast cancer progression. While the nuclear localization of maspin is essential for tumor suppression, we previously reported that the cytoplasmic localization of maspin was significantly correlated with poor prognosis in breast cancer patients. To understand the mechanisms that underlie oncogenic role of cytoplasmic maspin, we studied its biological function in breast cancer cell lines. Subcellular localization of maspin in MDA-MB-231 breast cancer cells was mainly detected in the cytoplasm, whereas in MCF10A mammary epithelial cells, maspin was present in both cytoplasm and nucleus. In MDA-MB-231 cells, maspin overexpression promoted cell proliferation and cell invasion, whereas maspin downregulation resulted in the opposite effect. Further, we observed that SRGN protein levels were increased in MDA-MB-231 cells stably overexpressing maspin. Finally, maspin overexpression in MDA-MB-231 cells resulted in the N-cadherin and epithelial mesenchymal transition (EMT)-related transcription factors upregulation, and TGFβ signaling pathway activation. These results suggested that cytoplasmic maspin enhances the invasive and metastatic potential in breast cancer cells with aggressive phenotype by inducing EMT via SRGN/TGFβ axis. This study demonstrated a novel biological function of cytoplasmic maspin in progression of breast cancer cells with an aggressive phenotype.