Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Sjögren Syndrome antigen B regulates LIN28-let-7 axis in Caenorhabditis elegans and human.

LIN28 protein and let-7 family micro RNAs (miRNAs) that are an evolutionarily conserved from nematodes to humans are the important regulators of developmental timing by dynamically interacting with each other. However, regulators of LIN28 remain largely elusive. Here, we show the evidences that Sjögren Syndrome antigen B (SSB) protein associates and cooperates with LIN28A and LIN28B, mammalian orthologues of Caenorhabditis elegans lin-28, proteins in the nucleus. Knockdown of SSB in HEK293 cell line resulted in the decrease of the amount of LIN28B mRNAs and proteins, and the increase of the level of mature let-7 miRNAs. Furthermore, RNA interference of ssb-1 gene, a worm SSB orthologue, was sufficient to cause a heterochronic defect in seam cells of C. elegans, recapitulating the phenotype of lin-28 downregulation. Collectively, we suggest that SSB is an important regulator for the LIN28-let-7 axis.

Long-term depletion of cereblon induces mitochondrial dysfunction in cancer cells.

Cereblon (CRBN) is a multi-functional protein that acts as a substrate receptor of the E3 ligase complex and a molecular chaperone. While CRBN is proposed to function in mitochondria, its specific roles are yet to be established. Here, we showed that knockdown of CRBN triggers oxidative stress and calcium overload in mitochondria, leading to disruption of mitochondrial membrane potential. Notably, long-term CRBN depletion using PROteolysis TArgeting Chimera (PROTAC) induced irreversible mitochondrial dysfunction, resulting in cell death. Our collective findings indicate that CRBN is required for mitochondrial homeostasis in cells. [BMB Reports 2021; 54(6): 305-310].

Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways.

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized ß-transducin repeat-containing protein 1 (ßTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.

Serine 389 phosphorylation of 3-phosphoinositide-dependent kinase 1 by UNC-51-like kinase 1 affects its ability to regulate Akt and p70 S6kinase.

Phosphorylation of the signaling component by protein kinase often leads to a kinase cascade or feedback loop. 3-Phosphoinositide- dependent kinase 1 (PDK1) signaling pathway diverges into various kinases including Akt and p70 S6 kinase (p70S6k). However, the PDK1 feedback mechanism remains elusive. Here, we demonstrated that UNC-51-like kinase (ULK1), an autophagy initiator kinase downstream of mechanistic target of rapamycin (mTOR), directly phosphorylated PDK1 on serine 389 at the linker region. Furthermore, our data showed that this phosphorylation affected the kinase activity of PDK1 toward downstream substrates. These results suggest a possible negative feedback loop between PDK1 and ULK1. [BMB Reports 2020; 53(7): 373-378].

Disordered region of cereblon is required for efficient degradation by proteolysis-targeting chimera.

Proteolysis targeting chimeras (PROTACs) are an emerging strategy for promoting targeted protein degradation by inducing the proximity between targeted proteins and E3 ubiquitin ligases. Although successful degradation of numerous proteins by PROTACs has been demonstrated, the elements that determine the degradability of PROTAC-targeted proteins have not yet been explored. In this study, we developed von Hippel-Lindau-Cereblon (VHL-CRBN) heterodimerizing PROTACs that induce the degradation of CRBN, but not VHL. A quantitative proteomic analysis further revealed that VHL-CRBN heterodimerizing PROTACs induced the degradation of CRBN, but not the well-known immunomodulatory drug (IMiD) neo-substrates, IKAROS family zinc finger 1 (IKZF1) and -3 (IZKF3). Moreover, truncation of disordered regions of CRBN and the androgen receptor (AR) attenuated their PROTAC-induced degradation, and attachment of the disordered region to stable CRBN or AR facilitated PROTAC-induced degradation. Thus, these results suggest that the intrinsically disordered region of targeted proteins is essential for efficient proteolysis, providing a novel criterion for choosing degradable protein targets.

cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis.

Although the ubiquitin-proteasome system is believed to play an important role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS), caused by mutations in Cu/Zn-superoxide dismutase 1 (SOD1), the mechanism of how mutant SOD1 protein is regulated in cells is still poorly understood. Here we have demonstrated that cellular inhibitor of apoptosis proteins (cIAPs) are specifically associated with FALS-linked mutant SOD1 (mSOD1) and that this interaction promotes the ubiquitin-dependent proteasomal degradation of mutant SOD1. By utilizing cumate inducible SOD1 cells, we also showed that knock-down or pharmacologic depletion of cIAPs leads to H2O2 induced cytotoxicity in mSOD1 expressing cells. Altogether, our results reveal a novel role of cIAPs in FALS-associated mutant SOD1 regulation.

Synergistic effect of two E2 ubiquitin conjugating enzymes in SCF(hFBH1) catalyzed polyubiquitination.

Ubiquitination is a post translational modification which mostly links with proteasome dependent protein degradation. This process has been known to play pivotal roles in the number of biological events including apoptosis, cell signaling, transcription and translation. Although the process of ubiquitination has been studied extensively, the mechanism of polyubiquitination by multi protein E3 ubiquitin ligase, SCF complex remains elusive. In the present study, we identified UbcH5a as a novel stimulating factor for poly-ubiquitination catalyzed by SCF(hFBH1) using biochemical fractionations and MALDI-TOF. Moreover, we showed that recombinant UbcH5a and Cdc34 synergistically stimulate SCF(hFBH1) catalyzed polyubiquitination in vitro. These data may provide an important cue to understand the mechanism how the SCF complex efficiently polyubiquitinates target substrates.

Mortality and real cause of death from the nonlesional intracerebral hemorrhage.

OBJECTIVE: The case fatality rate of nonlesional intracerebral hemorrhage (n-ICH) was high and not changed. Knowing the causes is important to their prevention; however, the reasons have not been studied. The aims of this study were to determine the cause of death, to improve the clinical outcomes. METHODS: We retrospectively analyzed consecutive cases of nonlesional intracerebral hemorrhage in a prospective stroke registry from January 2010 to December 2010. RESULTS: Among 174 patients (61.83±13.36, 28-90 years), 29 patients (16.7%) died during hospitalization. Most common cause of death was initial neurological damage (41.4%, 12/29). Seventeen patients who survived the initial damage may then develop various potentially fatal complications. Except for death due to the initial neurological sequelae, death associated with immobilization (such as pneumonia or thromboembolic complication) was the most common in eight cases (8/17, 47.1%). However, death due to early rebleeding was not common and occurred in only 2 cases (2/17, 11.8%). Age, initial Glasgow Coma Scale, and diabetes mellitus were statistically significant factors influencing mortality (p<0.05). CONCLUSION: Mortality of n-ICH is still high. Initial neurological damage is the most important factor; however, non-neurological medical complications are a large part of case fatality. Most cases of death of patients who survived from the first bleeding were due to complications of immobilization. These findings have implications for clinical practice and planning of clinical trials. In addition, future conduct of a randomized study will be necessary in order to evaluate the benefits of early mobilization for prevention of immobilization related complications.