Deubiquitinating enzyme USP39 promotes the growth and metastasis of gastric cancer cells by modulating the degradation of RNA-binding protein RBM39.

It has been revealed recently that the RNA-binding motif protein RBM39 is highly expressed in several cancers, which results in poor patient survival. However, how RBM39 is regulated in gastric cancer cells is unknown. Here, affinity purification-mass spectrometry and a biochemical screening are employed to identify the RBM39-interacting proteins and the deubiquitinating enzymes (DUBs) that regulate the RBM39 protein level. Integration of the data obtained from these two approaches uncovers USP39 as the potential DUB that regulates RBM39 stability. Bioinformatic analysis discloses that USP39 is increased in gastric cancer tissues and its elevation shortens the duration of overall survival for gastric cancer patients. Biochemical experiments verify that USP39 and RBM39 interact with each other and highly colocalize in the nucleus. Expression of USP39 elevates while USP39 knockdown attenuates the RBM39 protein level and their interaction regulates this modulation and their colocalization. Mechanistic studies reveal that USP39 reduces the K48-linked polyubiquitin chains on RBM39, thus enhancing its stability and increasing the protein level by preventing its proteasomal degradation. USP39 overexpression promotes while its knockdown attenuates the growth, colony formation, migration, and invasion of gastric cancer cells. Interestingly, overexpression of RBM39 partially restores the impact of USP39 depletion while RBM39 knockdown partially abolishes the effect of USP39 overexpression on the growth, colony formation, migration, and invasion of gastric cancer cells. Collectively, this work identifies the first DUB for RBM39 and elucidates the regulatory functions and the underlying mechanism, providing a possible alternative approach to suppressing RBM39 by inhibiting USP39 in cancer therapy.

The aryl sulfonamide indisulam inhibits gastric cancer cell migration by promoting the ubiquitination and degradation of the transcription factor ZEB1.

Gastric cancer is one of the cancers with high morbidity and mortality worldwide. The aryl sulfonamide indisulam inhibits the proliferation of several types of cancer cells through its function as a molecular glue to promote the ubiquitination and degradation of RNA-binding motif protein 39 (RBM39). However, it is unknown whether and how indisulam regulates the migration of cancer cells. In this work, using label-free quantitative proteomics, we discover that indisulam significantly attenuates N-cadherin, a marker for epithelial to mesenchymal transition and migration of cancer cells. Our bioinformatics analysis and biochemical experiments reveal that indisulam promotes the interaction between the zinc finger E-box-binding homeobox 1 (ZEB1), a transcription factor of N-cadherin, and DCAF15, a substrate receptor of CRL4 E3 ubiquitin ligase, and enhances ZEB1 ubiquitination and proteasomal degradation. In addition, our cell line-based experiments demonstrate that indisulam inhibits the migration of gastric cancer cells in a ZEB1-dependent manner. Analyses of patient samples and datasets in public databases reveal that tumor tissues from patients with gastric cancer express high ZEB1 mRNA and this high expression reduces patient survival rate. Finally, we show that treatment of gastric tumor samples with indisulam significantly reduces ZEB1 protein levels. Therefore, this work discloses a new mechanism by which indisulam inhibits the migration of gastric cancer cells, indicating that indisulam exhibits different biological functions through distinct signaling molecules.

Efficient Chemical Synthesis of Multi-Monoubiquitylated and Diubiquitylated Histones by the α-Halogen Ketone-Mediated Strategy.

The chemical synthesis of homogeneously ubiquitylated histones is a powerful approach to decipher histone ubiquitylation-dependent epigenetic regulation. Among the various methods, α-halogen ketone-mediated conjugation chemistry has recently been an attractive strategy to generate single-monoubiquitylated histones for biochemical and structural studies. Herein, we report the use of this strategy to prepare not only dual- and even triple-monoubiquitylated histones but also diubiquitin-modified histones. We were surprised to find that the synthetic efficiencies of multi-monoubiquitylated histones were comparable to those of single-monoubiquitylated ones, suggesting that this strategy is highly tolerant to the number of ubiquitin monomers installed onto histones. The facile generation of a series of single-, dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate the influence of ubiquitylation patterns on the binding of DNA methyltransferase 1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific conjugation chemistry to generate chemically defined histones for epigenetic studies.

Transcriptional regulation and post-translational modifications in the glycolytic pathway for targeted cancer therapy.

Cancer cells largely rely on aerobic glycolysis or the Warburg effect to generate essential biomolecules and energy for their rapid growth. The key modulators in glycolysis including glucose transporters and enzymes, e.g. hexokinase 2, enolase 1, pyruvate kinase M2, lactate dehydrogenase A, play indispensable roles in glucose uptake, glucose consumption, ATP generation, lactate production, etc. Transcriptional regulation and post-translational modifications (PTMs) of these critical modulators are important for signal transduction and metabolic reprogramming in the glycolytic pathway, which can provide energy advantages to cancer cell growth. In this review we recapitulate the recent advances in research on glycolytic modulators of cancer cells and analyze the strategies targeting these vital modulators including small-molecule inhibitors and microRNAs (miRNAs) for targeted cancer therapy. We focus on the regulation of the glycolytic pathway at the transcription level (e.g., hypoxia-inducible factor 1, c-MYC, p53, sine oculis homeobox homolog 1, N6-methyladenosine modification) and PTMs (including phosphorylation, methylation, acetylation, ubiquitination, etc.) of the key regulators in these processes. This review will provide a comprehensive understanding of the regulation of the key modulators in the glycolytic pathway and might shed light on the targeted cancer therapy at different molecular levels.

Preparation of UFM1-Derived Probes through Highly Optimized Total Chemical Synthesis.

Ufmylation is involved in various cellular processes and associated with many human diseases. The understanding of this modification relies on the use of customized UFM1-derived probes for activity-based profiling of its related enzymes. This study presents a highly optimized total chemical synthesis for the generation of diverse UFM1-derived probes including UFM1-PA, Biotin-UFM1-PA and UFM1-AMC, in which a UFM1 C-terminal valine hydrazide was readily prepared by hydrazide-based ligation and used as a versatile handle for the installation of enzyme-sensitive warheads and fluorescent reporters. The resulting probes display high reactivity and selectivity for UFM1-specific enzymes in cell lysates. This strategy facilitates the generation and diversity of the UFM1-derived toolkit that can be employed to profile UFM1-specific enzymes, thereby shining insights into the dynamics of ufmylation.

The Bre1/Rad6 machinery: writing the central histone ubiquitin mark on H2B and beyond.

Mono-ubiquitination on H2B (H2Bub1) is an evolutionarily conserved histone post-translational modification implicated in various important physiological processes including DNA replication, transcription activation, and DNA damage repair. The Bre1/Rad6 ubiquitination machinery is currently considered to be the sole writer of H2Bub1, but the mechanistic basis by which it operates is unclear. Recently, the RING-type E3 ligase Bre1 was proposed to associate with the E2 enzyme Rad6 through a novel interaction between Bre1 RBD (Rad6 binding domain) and Rad6; and the RING domain of Bre1 that is responsible for the nucleosomal acidic patch binding also interacts with Rad6 to stimulate its catalytic activity. Recent discoveries have yielded evidence for the phenomenon of liquid-liquid phase separation in the context of H2Bub1, and its regulation by other histone post-translational modifications. This review summarizes current knowledge about Bre1/Rad6-mediated H2B ubiquitination, including the physiological functions and the molecular basis for writing and regulation of this central histone ubiquitin mark. Possible models for the Bre1/Rad6 machinery bound to nucleosomes bearing different modifications in the writing step are also disclosed.

Insights on the biological functions and diverse regulation of RNA-binding protein 39 and their implication in human diseases.

RNA-binding protein 39 (RBM39) involves in pre-mRNA splicing and transcriptional regulation. RBM39 is dysregulated in many cancers and its upregulation enhances cancer cell proliferation. Recently, it has been discovered that aryl sulfonamides act as molecular glues to recruit RBM39 to the CRL4DCAF15 E3 ubiquitin ligase complex for its ubiquitination and proteasomal degradation. Therefore, various studies have focused on the degradation of RBM39 by aryl sulfonamides in the aim of finding new cancer therapeutics. These discoveries also attracted focus for thorough study on the biological functions of RBM39. RBM39 was found to regulate the splicing and transcription of genes mainly involved in pre-mRNA splicing, cell cycle regulation, DNA damage response, and metabolism, but the understanding of these regulations is still in its infancy. This article reviews the advances of the current literature and discusses the remaining key issues on the biological function and dynamic regulation of RBM39 at the post-translational level.