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Protein ubiquitination plays a pivotal role in protein homeostasis. Ubiquitination may regulate the stability, activity, protein–protein interaction, and localization of a protein. Ubiquitination is subject to regulation by two groups of counteracting enzymes, the E3 ubiquitin ligases and deubiquitinases. Consistently, deubiquitinases are involved in essentially all biological processes. OTUB1, an OTU-family deubiquitinase, is a critical regulator of development, cancer, DNA damage response, and immune response. OTUB1 antagonizes the ubiquitination of a wide-spectrum of proteins through at least two different mechanisms. Besides direct deubiquitination, OTUB1 can also inhibit ubiquitination by non-canonically blocking ubiquitin transfer from certain ubiquitin-conjugases (E2). In this review, we start with a general background of protein ubiquitination and deubiquitination. Next, we introduce the basic characteristics of OTUB1 and then elaborate on the updated biological functions of OTUB1. Afterwards, we discuss potential mechanisms underlying the versatility and specificity of OTUB1 functions. In the end, we discuss the perspective that OTUB1 can be a potential therapeutic target for cancer.

Abstract Prevalent dysregulation of epigenetic modifications plays a pivotal role in cancer. Targeting epigenetic abnormality is a new strategy for cancer therapy. Understanding how conventional oncogenic factors cause epigenetic abnormality is of great basic and translational value. O-GlcNAcylation is a protein modification which affects physiology and pathophysiology. In mammals, O-GlcNAcylation is catalyzed by one single enzyme OGT and removed by one single enzyme OGA. O-GlcNAcylation is affected by the availability of the donor, UDP-GlcNAc, generated by the serial enzymatic reactions in the hexoamine biogenesis pathway (HBP). O-GlcNAcylation regulates a wide spectrum of substrates including many proteins involved in epigenetic modification. Like epigenetic modifications, abnormality of O-GlcNAcylation is also common in cancer. Studies have revealed substantial impact on HBP enzymes and OGT/OGA by oncogenic signals. In this review, we will first summarize how oncogenic signals regulate HBP enzymes, OGT and OGA in cancer. We will then integrate this knowledge with the up to date understanding how O-GlcNAcylation regulates epigenetic machinery. With this, we propose a signal axis from oncogenic signals through O-GlcNAcylation dysregulation to epigenetic abnormality in cancer. Further elucidation of this axis will not only advance our understanding of cancer biology but also provide new revenues towards cancer therapy.

Type VI CRISPR effector Cas13d from Ruminococcus flavefaciens XPD3002 (RfxCas13d) is an RNA-guided RNA endonuclease. RfxCas13d has been harnessed to knockdown gene expression with high specificity in various systems including mammalian cells. While inducible knockdown is advantageous over constitutive knockdown in many scenarios, current inducible systems of RfxCas13d express CRISPR RNA and Cas13d separately. Such systems could be cumbersome to handle and may hamper the application of RfxCas13d in some scenarios. Here, we design an all-in-one Cas13d lentivirus vector which renders efficient and inducible knockdown in a doxycycline dosage-dependent manner. Furthermore, we find that Cas13d has a short half-life in mammalian cells. As a result, knockdown can be promptly reversed after doxycycline withdrawal. This vector is particularly useful for applications involving indispensable genes and/or in cells hard to transduce.

Summary: Histone lysine methylation functions at the interface of the extracellular environment and intracellular gene expression. DOT1L is a versatile histone H3K79 methyltransferase with a prominent role in MLL-fusion leukemia, yet little is known about how DOT1L responds to extracellular stimuli. Here, we report that DOT1L protein stability is regulated by the extracellular glucose level through the hexosamine biosynthetic pathway (HBP). Mechanistically, DOT1L is O-GlcNAcylated at evolutionarily conserved S1511 in its C terminus. We identify UBE3C as a DOT1L E3 ubiquitin ligase promoting DOT1L degradation whose interaction with DOT1L is susceptible to O-GlcNAcylation. Consequently, HBP enhances H3K79 methylation and expression of critical DOT1L target genes such as HOXA9/MEIS1, promoting cell proliferation in MLL-fusion leukemia. Inhibiting HBP or O-GlcNAc transferase (OGT) increases cellular sensitivity to DOT1L inhibitor. Overall, our work uncovers O-GlcNAcylation and UBE3C as critical determinants of DOT1L protein abundance, revealing a mechanism by which glucose metabolism affects malignancy progression through histone methylation.

The development of neonatal necrotising enterocolitis has been temporally associated with red blood cell transfusions in retrospective human studies. Here, the authors develop a neonatal mouse model of necrotising enterocolitis in anaemic mice receiving red blood cell transfusion that recapitulates features of the human condition.

Autophagy plays a pivotal role in physiology and pathophysiology, including cancer. Mechanisms of autophagy dysregulation in cancer remain elusive. Loss of function of TRIM28, a multifunction protein, is seen in familial kidney malignancy, but the mechanism by which TRIM28 contributes to the etiology of kidney malignancy is unclear. In this study, we show TRIM28 retards kidney cancer cell proliferation through inhibiting autophagy. Mechanistically, we find TRIM28 promotes ubiquitination and proteasome-mediated degradation of transcription factor TFE3, which is critical for autophagic gene expression. Genetic activation of TFE3 due to gene fusion is known to cause human kidney malignancy, but whether and how transcription activation by TFE3 involves chromatin changes is unclear. Here, we find another mode of TFE3 activation in human renal carcinoma. We find that TFE3 is constitutively localized to the cell nucleus in human and mouse kidney cancer, where it increases autophagic gene expression and promotes cell autophagy as well as proliferation. We further uncover that TFE3 interacts with and recruits histone H3K27 demethylase KDM6A for autophagic gene upregulation. We reveal that KDM6A contributes to expression of TFE3 target genes through increasing H3K4me3 rather than demethylating H3K27. Collectively, in this study, we identify a functional TRIM28-TFE3-KDM6A signal axis, which plays a critical role in kidney cancer cell autophagy and proliferation. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT-DBD interaction. [ABSTRACT FROM AUTHOR]

Missense p53 mutants often accumulate in tumors and drive progression through gain-of-function. MDM2 efficiently degrades wild type p53, but fails to degrade mutant p53 in tumor cells. Previous studies revealed that mutant p53 inhibits MDM2 auto-ubiquitination, suggesting that the interaction inhibits MDM2 E3 activity. Recent work showed that MDM2 E3 activity is stimulated by intramolecular interaction between the RING and acidic domains. Here we show that in the mutant p53-MDM2 complex, the mutant p53 core domain binds to the MDM2 acidic domain with significantly higher avidity compared to wild type p53. Mutant p53-MDM2 complex is deficient in catalyzing ubiquitin release from the activated E2 conjugating enzyme. An MDM2 construct with extra copies of the acidic domain is resistant to inhibition by mutant p53, and efficiently promotes mutant p53 ubiquitination and degradation. The results suggest that mutant p53 interferes with the intramolecular auto-activation mechanism of MDM2, contributing to reduced ubiquitination and increased accumulation in tumor cells. [ABSTRACT FROM AUTHOR]

The MDMX oncoprotein is an important regulator of tumor suppressor p53 activity during embryonic development. Despite sequence homology to the ubiquitin E3 ligase MDM2, MDMX depletion activates p53 without significant increase in p53 level, implicating a degradation-independent mechanism. We present evidence that MDMX inhibits the sequence-specific DNA binding activity of p53. This function requires the cooperation between MDMX and CK1α, and phosphorylation of S289 on MDMX. Depletion of MDMX or CK1α increases p53 DNA binding without stabilization of p53. A proteolytic fragment release assay revealed that in the MDMX-p53 complex, the MDMX acidic domain and RING domain interact stably with the p53 DNA binding domain. These interactions are referred to as secondary interactions because they only occur after the canonical-specific binding between the MDMX and p53 N termini, but exhibit significant binding stability in the mature complex. CK1α cooperates with MDMX to inhibit p53 DNA binding by further stabilizing the MDMX acidic domain and p53 core domain interaction. These results suggest that secondary intermolecular interaction is important in p53 regulation by MDMX, which may represent a common phenomenon in complexes containing multidomain proteins. [ABSTRACT FROM AUTHOR]

The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels. [ABSTRACT FROM AUTHOR]

Nucleomethylin (NML), a novel nucleolar protein, is important for mediating the assembly of the energy-dependent nucleolar silencing complex (eNoSC), which also contains SirT1 and SUV39H1. eNoSC represses rRNA transcription during nutrient deprivation, thus reducing energy expenditure and improving cell survival. We found that NML is an RNA binding protein that copurifies with 5S, 5.8S, and 28S rRNA. The SirT1 and RNA binding regions on NML showed partial overlap, and the NML-SirT1 interaction was competitively inhibited by rRNA. Nutrient deprivation triggered downregulation of rRNA transcription, reduced the level of NML-associated rRNA, and stimulated NML-SirT1 binding. Assembly of eNoSC facilitated repression of prerRNA transcription. These results suggest that nascent rRNA generates a positive-feedback signal by suppressing the assembly of eNoSC and protecting active ribosomal DNA units from heterochromatin formation. This RNA-mediated mechanism enables the eNoSC to amplify the effects of upstream nutrient-responsive regulators. [ABSTRACT FROM AUTHOR]

The chromosomal region encoding the nuclear NAD+ synthesis enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT1) is frequently deleted in human cancer. We describe evidence that NMNAT1 interacts with the nucleolar repressor protein nucleomethylin and is involved in regulating rRNA transcription. NMNAT1 binds to nucleomethylin and is recruited into a ternary complex containing the NAD+-dependent deacetylase SirT1. NMNAT1 expression stimulates the deacetylase function of SirT1. Knockdown of NMNAT1 enhances rRNA transcription and promotes cell death after nutrient deprivation. Furthermore, NMNAT1 expression is induced by DNA damage and plays a role in preventing cell death after damage. Heterozygous deletion of NMNAT1 in lung tumor cell lines correlates with low expression level and increased sensitivity to DNAdamage. These results suggest that NMNAT1 deletion in tumors may contribute to transformation by increasing rRNA synthesis, but may also increase sensitivity to nutrient stress and DNA damage. [ABSTRACT FROM AUTHOR]

Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs. [ABSTRACT FROM AUTHOR]

Higher demand for nutrients including glucose is characteristic of cancer. "Starving cancer" has been pursued to curb tumor progression. An intriguing regime is to inhibit glucose transporter GLUT1 in cancer cells. In addition, during cancer progression, cancer cells may suffer from insufficient glucose supply. Yet, cancer cells can somehow tolerate glucose starvation. Uncovering the underlying mechanisms shall shed insight into cancer progression and benefit cancer therapy. TFE3 is a transcription factor known to activate autophagic genes. Physiological TFE3 activity is regulated by phosphorylationtriggered translocation responsive to nutrient status. We recently reported TFE3 constitutively localizes to the cell nucleus and promotes cell proliferation in kidney cancer even under nutrient replete condition. It remains unclear whether and how TFE3 responds to glucose starvation. In this study, we show TFE3 promotes kidney cancer cell resistance to glucose starvation by exposing cells to physiologically relevant glucose concentration. We find glucose starvation triggers TFE3 protein stabilization through increasing its O-GlcNAcylation. Furthermore, through an unbiased functional genomic study, we identify SLC36A1, a lysosomal amino acid transporter, as a TFE3 target gene sensitive to TFE3 protein level. We find SLC36A1 is overexpressed in kidney cancer, which promotes mTOR activity and kidney cancer cell proliferation. Importantly, SLC36A1 level is induced by glucose starvation through TFE3, which enhances cellular resistance to glucose starvation. Suppressing TFE3 or SLC36A1 significantly increases cellular sensitivity to GLUT1 inhibitor in kidney cancer cells. Collectively, we uncover a functional TFE3–SLC36A1 axis that responds to glucose starvation and enhances starvation tolerance in kidney cancer. [ABSTRACT FROM AUTHOR]

Prostate cancer is a leading cause of cancer-related mortality in males. Dysregulation of RNA adenine N-6 methylation (m6A) contributes to cancer malignancy. m6A on mRNA may affect mRNA splicing, turnover, transportation, and translation. m6A exerts these effects, at least partly, through dedicated m6A reader proteins, including YTH domain--containing family protein 2 (YTHDF2). YTHDF2 is necessary for development while its dysregulation is seen in various cancers, including prostate cancer. However, the mechanism underlying the dysregulation and function of YTHDF2 in cancer remains elusive. Here, we find that the deubiquitinase OUT domain--containing ubiquitin aldehyde-binding protein 1 (OTUB1) increases YTHDF2 protein stability by inhibiting its ubiquitination. With in vivo and in vitro ubiquitination assays, OTUB1 is shown to block ubiquitin transfer to YTHDF2 independent of its deubiquitinase activity. Furthermore, analysis of functional transcriptomic data and m6A-sequencing data identifies PRSS8 as a potential tumor suppressor gene. OTUB1 and YTHDF2 decrease mRNA and protein levels of PRSS8, which is a trypsinlike serine protease. Mechanistically, YTHDF2 binds PRSS8 mRNA and promotes its degradation in an m6A-dependent manner. Further functional study on cellular and mouse models reveals PRSS8 is a critical downstream effector of the OTUB1--YTHDF2 axis in prostate cancer. We find in prostate cancer cells, PRSS8 decreases nuclear β-catenin level through E-cadherin, which is independent of its protease activity. Collectively, our study uncovers a key regulator of YTHDF2 protein stability and establishes a functional OTUB1-- YTHDF2--PRSS8 axis in prostate cancer. [ABSTRACT FROM AUTHOR]

Liver cancer is notoriously refractory to conventional therapeutics. Tumor progression is governed by the interplay between tumor-promoting genes and tumor-suppressor genes. BRD4, an acetyl lysine-binding protein, is overexpressed in many cancer types, which promotes activation of a pro-tumor gene network. But the underlying mechanism for BRD4 overexpression remains incompletely understood. In addition, understanding the regulatory mechanism of BRD4 protein level will shed insight into BRD4-targeting therapeutics. In this study, we investigated the potential relation between BRD4 protein level and P53, the most frequently dysregulated tumor suppressor. By analyzing the TCGA datasets, we first identify a strong negative correlation between protein levels of P53 and BRD4 in liver cancer. Further investigation shows that P53 promotes BRD4 protein degradation. Mechanistically, P53 indirectly represses the transcription of USP1, a deubiquitinase, through the P21-RB1 axis. USP1 itself is also overexpressed in liver cancer and we show USP1 deubiquitinates BRD4 in vivo and in vitro, which increases BRD4 stability. With cell proliferation assays and xenograft model, we show the protumor role of USP1 is partially mediated by BRD4. With functional transcriptomic analysis, we find the USP1-BRD4 axis upholds expression of a group of cancer-related genes. In summary, we identify a functional P53-P21-RB1-USP1-BRD4 axis in liver cancer. [ABSTRACT FROM AUTHOR]

Advanced hepatocellular carcinoma (HCC) has a dismal prognosis. KDM1A (lysine demethylase 1A), overexpressed in multiple cancer types, is a lysine demethylase that targets both histone and nonhistone proteins. However, it is unclear how KDM1A expression affects HCC etiology. Here, we show that KDM1A can interact with and demethylate FKBP8 (FKBP prolyl isomerase 8), a cytoplasmic protein that regulates cell survival through the antiapoptotic protein BCL2 (B-cell lymphoma-2). We show that demethylation of FKBP8 enhances its ability to stabilize BCL2. Consistently, we observed positive correlation between KDM1A and BCL2 protein levels in liver cancer patients. Functionally, we reveal that FKBP8 demethylation by KDM1A is critical for liver cancer cell growth in vitro and in vivo. We went on to explore the mechanisms that might regulate KDM1A cytoplasmic localization. We found that the cytoplasmic localization and protein stability of KDM1A were promoted by acetylation at lysine-117 by the acetyl transferase KAT8 (lysine acetyltransferase 8). In agreement with this, we show that KDM1A--K117 (lysine 117) acetylation promotes demethylation of FKBP8 and level of BCL2. Finally, it has been shown that the efficacy of sorafenib, a first-line treatment for advanced HCC, is limited by clinical resistance. We show that KDM1A and BCL2 protein levels are increased during acquired sorafenib resistance, whereas inhibiting KDM1A can antagonize sorafenib resistance. Collectively, these results define a functional KDM1A--FKBP8--BCL2 axis in HCC. [ABSTRACT FROM AUTHOR]

Distinct lysine methylation marks on histones create dynamic signatures deciphered by the "effector" modules, although the underlying mechanisms remain unclear. We identified the plant homeodomainand Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the "cross-talk" of histone modifications. [ABSTRACT FROM AUTHOR]