Hudan Liu

The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases

We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3′ to 5′ decay of an mRNA. The association of DcpS with 3′ to 5′ exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay‐dependent decapping pathway. We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity. Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA. Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues. Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function.

Hsp90 regulates processing of NF-κB2 p100 involving protection of NF-κB-inducing kinase (NIK) from autophagy-mediated degradation

NF-κB-inducing kinase (NIK) is required for NF-κB activation based on the processing of NF-κB2 p100. Here we report a novel mechanism of NIK regulation involving the chaperone 90 kDa heat shock protein (Hsp90) and autophagy. Functional inhibition of Hsp90 by the anti-tumor agent geldanamycin (GA) efficiently disrupts its interaction with NIK, resulting in NIK degradation and subsequent blockage of p100 processing. Surprisingly, GA-induced NIK degradation is mediated by autophagy, but largely independent of the ubiquitin-proteasome system. Hsp90 seems to be specifically involved in the folding/stabilization of NIK protein, because GA inhibition does not affect NIK mRNA transcription and translation. Furthermore, Hsp90 is not required for NIK-mediated recruitment of the α subunit of IκB kinase to p100, a key step in induction of p100 processing. These findings define an alternative mechanism for Hsp90 client degradation and identify a novel function of autophagy in NF-κB regulation. These findings also suggest a new therapeutic strategy for diseases associated with p100 processing.

Scavenger Decapping Activity Facilitates 5′ to 3′ mRNA Decay

ABSTRACT mRNA degradation occurs through distinct pathways, one primarily from the 5′ end of the mRNA and the second from the 3′ end. Decay from the 3′ end generates the m7GpppN cap dinucleotide, which is subsequently hydrolyzed to m7Gp and ppN in Saccharomyces cerevisiae by a scavenger decapping activity termed Dcs1p. Although Dcs1p functions in the last step of mRNA turnover, we demonstrate that its activity modulates earlier steps of mRNA decay. Disruption of the DCS1 gene manifests a threefold increase of the TIF51A mRNA half-life. Interestingly, the hydrolytic activity of Dcs1p was essential for the altered mRNA turnover, as Dcs1p, but not a catalytically inactive Dcs1p mutant, complemented the increased mRNA stability. Mechanistic analysis revealed that 5′ to 3′ exoribonucleolytic activity was impeded in the dcs1Δ strain, resulting in the accumulation of uncapped mRNA. These data define a new role for the Dcs1p scavenger decapping enzyme and demonstrate a novel mechanism whereby the final step in the 3′ mRNA decay pathway can influence 5′ to 3′ exoribonucleolytic activity.

DcpS scavenger decapping enzyme can modulate pre-mRNA splicing.

The human scavenger decapping enzyme, DcpS, functions to hydrolyze the resulting cap structure following cytoplasmic mRNA decay yet is, surprisingly, a nuclear protein by immunofluorescence. Here, we show that DcpS is a nucleocytoplasmic shuttling protein that contains separable nuclear import and Crm-1-dependent export signals. We postulated that the presence of DcpS in both cellular compartments and its ability to hydrolyze cap structure may impact other cellular events dependent on cap-binding proteins. An shRNA-engineered cell line with markedly diminished DcpS levels led to a corresponding reduction in cap-proximal intron splicing of a reporter minigene and endogenous genes. The impaired cap catabolism and resultant imbalanced cap concentrations were postulated to sequester the cap-binding complex (CBC) from its normal splicing function. In support of this explanation, DcpS efficiently displaced the nuclear cap-binding protein Cbp20 from cap structure, and complementation with Cbp20 reversed the reduced splicing, indicating that modulation of splicing by DcpS is mediated through Cbp20. Our studies demonstrate that the significance of DcpS extends beyond its well-characterized role in mRNA decay and involves a broader range of functions in RNA processing including nuclear pre-mRNA splicing.

Myc promotes glutaminolysis in human neuroblastoma through direct activation of glutaminase 2

Deamidation of glutamine to glutamate by glutaminase 1 (GLS1, also called GLS) and GLS2 is an essential step in both glutaminolysis and glutathione (GSH) biosynthesis. However, mechanisms whereby cancer cells regulate glutamine catabolism remains largely unknown. We report here that N-Myc, an essential Myc family member, promotes conversion of glutamine to glutamate in MYCN-amplified neuroblastoma cells by directly activating GLS2, but not GLS1, transcription. Abrogation of GLS2 function profoundly inhibited glutaminolysis, which resulted in feedback inhibition of aerobic glycolysis likely due to thioredoxin-interacting protein (TXNIP) activation, dramatically decreasing cell proliferation and survival in vitro and in vivo. Moreover, elevated GLS2 expression is significantly elevated in MYCN-amplified neuroblastomas in comparison with non-amplified ones, correlating with unfavorable patient survival. In aggregate, these results reveal a novel mechanism deciphering context-dependent regulation of metabolic heterogeneities, uncovering a previously unsuspected link between Myc, GLS2 and tumor metabolism.

Critical roles of NOTCH1 in acute T-cell lymphoblastic leukemia

NOTCH1 plays a central role in T-cell development and, when aberrantly activated, in acute T-cell lymphoblastic leukemia (T-ALL). As a transmembrane receptor that is ultimately converted into a transcription factor, NOTCH1 directly activates a spectrum of target genes, which function to mediate NOTCH1 signaling in normal or transformed T cells. During physiologic T-cell development, NOTCH1 has important functions in cell fate determination, proliferation, survival and metabolism. Activating NOTCH1 mutations occur in more than half of human patients with T-ALL, suggesting an important role for aberrant NOTCH1 signaling in the pathogenesis of this disease. Inhibiting NOTCH1 signaling in patient-derived cell lines and murine T-ALLs leads to growth arrest and/or apoptosis suggesting that NOTCH1 inhibitors can improve T-ALL treatment. However, there are challenges to translate NOTCH1 inhibitors to the clinic because of toxicity and resistance. This review focuses on molecular mechanisms of oncogenic NOTCH1 signaling, molecular and functional analysis of NOTCH1 transcriptional targets in T-ALL, and recent advances in therapeutic targeting of NOTCH1.

Cloning and characterization of a mouse liver-specific gene mfrep-1, upregulated in liver regeneration

ABSTRACTHuman fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-1/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-1/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-1/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice. mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced expression of mfrep-1 maintained high until 72 h and then declined slowly to the basal level. Immunohistochemistry assessment confirmed the up-regulated expression of MFREP-1 protein in parenchymal cells during liver regeneration. These data suggested that MFREP-1 might play an important role in liver regeneration and be involved in the regulation of cell growth.Genbank accession number: The cDNA sequence reported here was deposited in GenBank. MFREP-1, AF478470.

HIV-1 Rev–binding protein accelerates cellular uptake of iron to drive Notch-induced T cell leukemogenesis in mice

Somatic activating mutations in Notch1 contribute to the pathogenesis of T cell acute lymphoblastic lymphoma (T-ALL), but how activated Notch1 signaling exerts this oncogenic effect is not completely understood. Here we identify HIV-1 Rev-binding protein (Hrb), a component of the clathrin-mediated endocytosis machinery, as a critical mediator of Notch-induced T-ALL development in mice. Hrb was found to be a direct transcriptional target of Notch1, and Hrb loss reduced the incidence or delayed the onset of T-ALL in mouse models in which activated Notch1 signaling either contributes to or drives leukemogenesis. Consistent with this observation, Hrb supported survival and proliferation of hematopoietic and T cell precursor cells in vitro. We demonstrated that Hrb accelerated the uptake of transferrin, which was required for upregulation of the T cell protooncogene p21. Indeed, iron-deficient mice developed Notch1-induced T-ALL substantially more slowly than control mice, further supporting a critical role for iron uptake during leukemogenesis. Taken together, these results reveal that Hrb is a critical Notch target gene that mediates lymphoblast transformation and disease progression via its ability to satisfy the enhanced demands of transformed lymphoblasts for iron. Further, our data suggest that Hrb may be targeted to improve current treatment or design novel therapies for human T-ALL patients.

Targeting oncogenic Myc as a strategy for cancer treatment

The MYC family oncogene is deregulated in >50% of human cancers, and this deregulation is frequently associated with poor prognosis and unfavorable patient survival. Myc has a central role in almost every aspect of the oncogenic process, orchestrating proliferation, apoptosis, differentiation, and metabolism. Although Myc inhibition would be a powerful approach for the treatment of many types of cancers, direct targeting of Myc has been a challenge for decades owing to its “undruggable” protein structure. Hence, alternatives to Myc blockade have been widely explored to achieve desirable anti-tumor effects, including Myc/Max complex disruption, MYC transcription and/or translation inhibition, and Myc destabilization as well as the synthetic lethality associated with Myc overexpression. In this review, we summarize the latest advances in targeting oncogenic Myc, particularly for cancer therapeutic purposes.Cancer: Therapeutic strategies target myc from all directionsThe cancer gene myc is the subject of a variety of research strategies aimed at developing anti-cancer therapies. Myc, which regulates at least 15% of all genes including those involved in cell development and survival, is mutated and overexpressed in many cancer types. While inhibiting myc is an obvious anti-cancer strategy, directly targeting myc has proved difficult. Dr Guoliang Qing and colleagues from Wuhan University have reviewed advances in myc-targeting strategies as potential cancer therapies. Current strategies include indirectly targeting myc by inhibiting its regulation and stability, and interaction with its partner Max, which is necessary for its regulation of gene expression. Other strategies involve inducing a mutation in a gene that interacts with myc to kill cancer cells. These and future innovative approaches provide promise for effective therapeutics against myc-dependent cancers.

Stabilization of Notch1 by the Hsp90 Chaperone is Crucial for T-Cell Leukemogenesis

Purpose: Notch1 deregulation is assuming a focal role in T-cell acute lymphoblastic leukemia (T-ALL). Despite tremendous advances in our understanding of Notch1 transcriptional programs, the mechanisms by which Notch1 stability and turnover are regulated remain obscure. The goal of the current study is to identify intracellular Notch1 (ICN1, the activated form of Notch1) binding partner(s) regulating its stability and activity. Experimental Design: We employed immunoaffinity purification to identify ICN1-associating partner(s) and used coimmunoprecipitation to verify the endogenous protein interaction. Pharmacologic or short hairpin RNA–mediated inhibition was applied in loss-of-function assays to assess the role of tentative binding partner(s) in modulating ICN1 protein stability as well as affecting T-ALL cell expansion in vitro and in vivo. Mechanistic analysis involved protein degradation and polyubiquitination assays. Results: We identify the Hsp90 chaperone as a direct ICN1-binding partner essential for its stabilization and transcriptional activity. T-ALL cells exhibit constitutive endogenous ICN1–Hsp90 interaction and Hsp90 depletion markedly decreases ICN1 levels. The Hsp90-associated E3 ubiquitin ligase Stub1 mediates the ensuring proteasome-dependent ICN1 degradation. Administration of 17-AAG or PU-H71, two distinct Hsp90 inhibitors, depletes ICN1, inhibits T-ALL cell proliferation, and triggers dramatic apoptotic cell death. Systemic treatment with PU-H71 reduces ICN1 expression and profoundly inhibits murine T-ALL allografts as well as human T-ALL xenografts. Conclusions: Our findings demonstrate Hsp90 blockade leads to ICN1 destabilization, providing an alternative strategy to antagonize oncogenic Notch1 signaling with Hsp90-selective inhibitors. Clin Cancer Res; 23(14); 3834–46. ©2017 AACR.