Explore the vast range of titles available.

Impaired turnover of the autophagy substrate p62 leads to liver injury. p62 inhibits the ubiquitin ligase Keap1, leading to stabilization of the transcription factor Nrf2. High levels of p62 in autophagy deficient animals leads to unusually high expression of Nrf2 targets genes and results in liver injury. Impaired selective turnover of p62 by autophagy causes severe liver injury accompanied by the formation of p62-positive inclusions and upregulation of detoxifying enzymes. These phenotypes correspond closely to the pathological conditions seen in human liver diseases, including alcoholic hepatitis and hepatocellular carcinoma. However, the molecular mechanisms and pathophysiological processes in these events are still unknown. Here we report the identification of a novel regulatory mechanism by p62 of the transcription factor Nrf2, whose target genes include antioxidant proteins and detoxification enzymes. p62 interacts with the Nrf2-binding site on Keap1, a component of Cullin-3-type ubiquitin ligase for Nrf2. Thus, an overproduction of p62 or a deficiency in autophagy competes with the interaction between Nrf2 and Keap1, resulting in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Our findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.

Previously we have reported that stratifin (SFN, 14-3-3 sigma) acts as a novel oncogene, accelerating the tumor initiation and progression of lung adenocarcinoma. Here, pull-down assay and LC-MS/MS analysis revealed that ubiquitin-specific protease 8 (USP8) specifically bound to SFN in lung adenocarcinoma cells. Both USP8 and SFN showed higher expression in human lung adenocarcinoma than in normal lung tissue, and USP8 expression was significantly correlated with SFN expression. Expression of SFN, but not of USP8, was associated with histological subtype, pathological stage, and poor prognosis. USP8 stabilizes receptor tyrosine kinases (RTKs) such as EGFR and MET by deubiquitination, contributing to the proliferative activity of many human cancers including non-small cell lung cancer. In vitro, USP8 binds to SFN and they co-localize at the early endosomes in lung adenocarcinoma cells. Moreover, USP8 or SFN knockdown leads to downregulation of tumor cellular proliferation and upregulation of apoptosis, p-EGFR or p-MET, which are related to the degradation pathway, and accumulation of ubiquitinated RTKs, leading to lysosomal degradation. Additionally, mutant USP8, which is unable to bind to SFN, reduces the expression of RTKs and p-STAT3. We also found that interaction with SFN is critical for USP8 to exert its autodeubiquitination function and avoid dephosphorylation by PP1. Our findings demonstrate that SFN enhances RTK stabilization through abnormal USP8 regulation in lung adenocarcinoma, suggesting that SFN could be a more suitable therapeutic target for lung adenocarcinoma than USP8.

DNA double-strand breaks (DSBs) pose a potent threat to genome integrity. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DSBs elicit a signalling cascade that modifies the chromatin surrounding the break, first by ATM-dependent phosphorylation and then by RNF8-, RNF168- and BRCA1-dependent regulatory ubiquitination. Here we report that OTUB1, a deubiquitinating enzyme, is an inhibitor of DSB-induced chromatin ubiquitination. Surprisingly, we found that OTUB1 suppresses RNF168-dependent poly-ubiquitination independently of its catalytic activity. OTUB1 does so by binding to and inhibiting UBC13 (also known as UBE2N), the cognate E2 enzyme for RNF168. This unusual mode of regulation is unlikely to be limited to UBC13 because analysis of OTUB1-associated proteins revealed that OTUB1 binds to E2s of the UBE2D and UBE2E subfamilies. Finally, OTUB1 depletion mitigates the DSB repair defect associated with defective ATM signalling, indicating that pharmacological targeting of the OTUB1–UBC13 interaction might enhance the DNA damage response. Enhanced DNA damage response When double-strand breaks occur in eukaryotic DNA, the proteinaceous chromatin structure that protects and organizes the genome must be removed from around the break to allow access by the repair factors. Such chromatin displacement involves the addition of ubiquitin moieties to histones near the break by the E3 ubiquitin ligases RNF8 and RNF168. Nakada et al . have now identified OTUB1, a deubiquitinating enzyme of the ovarian tumour (OTU) family, as an inhibitor of double-strand break-induced chromatin ubiquitination. Acting in a non-canonical, non-catalytic manner, it inhibits the E2 enzyme that functions upstream of RNF168, and potentially other E2s. OTUB1 depletion mitigates the DNA-repair defect associated with defective ATM (ataxia telangiectasia mutated) signalling, indicating that pharmacological targeting of the OTUB1–UBC13 interaction might enhance the DNA damage response. When double-strand breaks occur in eukaryotic DNA, the chromatin that protects and organizes the genome must be removed from the vicinity of the break to allow repair factors to bind. Such chromatin displacement involves the addition of ubiquitin groups to histone proteins near the break by the ubiquitin ligases RNF8 and RNF168. Here it is shown that the enzyme OTUB1 prevents RNF168-dependent poly-ubiquitination. Pharmacological targeting of this process might enhance the DNA damage response.

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It is composed of one catalytic 20S proteasome and two 19S regulatory particles attached on both ends of 20S proteasomes. Here, we describe the identification of Adrm1 as a novel proteasome interacting protein in mammalian cells. Although the overall sequence of Adrm1 has weak homology with the yeast Rpn13, the amino‐ and carboxyl‐terminal regions exhibit significant homology. Therefore, we designated it as hRpn13. hRpn13 interacts with a base subunit Rpn2 via its amino‐terminus. The majority of 26S proteasomes contain hRpn13, but a portion of them does not, indicating that hRpn13 is not an integral subunit. Intriguingly, we found that hRpn13 recruits UCH37, a deubiquitinating enzyme known to associate with 26 proteasomes. The carboxyl‐terminal regions containing KEKE motifs of both hRpn13 and UCH37 are involved in their physical interaction. Knockdown of hRpn13 caused no obvious proteolytic defect but loss of UCH37 proteins and decrease in deubiquitinating activity of 26S proteasomes. Our results indicate that hRpn13 is essential for the activity of UCH37.

Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1- dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.

Sister chromatid cohesion, mediated by a complex called cohesin, is crucial—particularly at centromeres—for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro , suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.

Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation. Yes-associated protein (YAP) is a growth-promoting transcription co-activator that regulates the malignancy of various cancers, however its regulation is not fully understood. Here the authors show that phosphatdylserine at recycling endosomes regulates YAP signalling pathway.

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

The DNA-binding chromodomain helicase CHD8 regulates gene expression but how it acts on specific genes has been unclear. During early embryogenesis in mice, CHD8 recruits histone H1 to the p53-dependent promotors of apoptotic genes and thereby prevents massive cell death at this stage of development. The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8 −/− mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1.

Calpain-10 (CAPN10) is the calpain family protease identified as the first candidate susceptibility gene for type 2 diabetes mellitus (T2DM). However, the detailed molecular mechanism has not yet been elucidated. Here we report that CAPN10 processes microtubule associated protein 1 (MAP1) family proteins into heavy and light chains and regulates their binding activities to microtubules and actin filaments. Immunofluorescent analysis of Capn10 −/− mouse embryonic fibroblasts shows that MAP1B, a member of the MAP1 family of proteins, is localized at actin filaments rather than at microtubules. Furthermore, fluorescence recovery after photo-bleaching analysis shows that calpain-10 regulates actin dynamics via MAP1B cleavage. Moreover, in pancreatic islets from CAPN10 knockout mice, insulin secretion was significantly increased both at the high and low glucose levels. These findings indicate that deficiency of calpain-10 expression may affect insulin secretion by abnormal actin reorganization, coordination and dynamics through MAP1 family processing.