Aberrant activation of RPB1 is critical for cell overgrowth in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a group of highly aggressive malignancies with a 5-year overall survival of less than 40%. Cell overgrowth with defective apoptosis is a hallmark of AML, but little is known about how it occurs. Here, we show that aberrant activation of the largest subunit of RNA polymerase II (RPB1) encoded by POLR2A gene is critically involved in this hallmark. We retrospectively analyzed the expression profiles of POLR2A and RPB1 in a panel of AML cell lines, primary AML patients and peripheral blood samples. Meanwhile, correlation analysis was used to explore the correlation between the expression of RPB1 with tumor burden and overall survival time in untreated AML samples. RNA-Seq approach was performed to identify the differentially expressed genes between RPB1 silencing AML cells with control cells after knocking out RPB1. Furthermore, orthotopic AML models were established with RPB1 silencing and control cells to investigate the effects of RPB1 protein level on leukemia cell growth. In most AML patients, RPB1 was aberrantly activated and closely associated with poor prognosis, but not in normal hematopoietic cells. Global transcriptomic analysis revealed that POLR2A knockout strongly impaired growth of AML cells by selectively depleting a substantial set of AML-related oncogenic and anti-apoptosis genes such as MYC, RUNX2, MEIS1, CDC25A and BCL-2. Silencing RPB1 by genetic technology led to a potent regression of human refractory AML in mouse models. These findings reveal that dysregulated RPB1 is a central oncogenic hub that drives overgrowth by hijacking an array of oncogenic and anti-apoptosis factors. Targeting RPB1 is a potential therapeutic for treating AML.

Identification of the UBA2-WTIP fusion gene in acute myeloid leukemia.

Identifying and targeting oncogenic fusion genes have revolutionized the treatment of leukemia, such as PML-RARα fusion gene in acute promyelocytic leukemia. Here we identified an intrachromosomal fusion gene located on chromosome 19q.13 between UBA2 and WTIP gene in a case of acute myeloid leukemia. The UBA2-WTIP fusion gene contains the N-terminal E1_enzyme_family, VAE_Ubl domains of UBA2, and the C-terminal LIM domains of WTIP. The UBA2-WTIP fusion was detected by reverse transcriptase polymerase chain reaction and Sanger sequencing in 19 of 56 acute myeloid leukemia samples (33.9%). Ectopic expression of the UBA2-WTIP fusion in human acute myeloid leukemia KG-1a cells showed enhanced cell proliferation both in vitro and in vivo. The UBA2-WTIP fusion induced phosphorylation of STAT3, STAT5 and ERK1/2, and abrogates WTIP-mediated mammalian processing body formation. Finally, triptolide displayed selective cytotoxicity against KG-1a cells harboring the UBA2-WTIP fusion. Collectively, our findings suggest that the UBA2-WTIP fusion is an oncogenic fusion gene, as well as a promising therapeutic target for the treatment of acute myeloid leukemia.

Novel synthetic 4-chlorobenzoyl berbamine inhibits c-Myc expression and induces apoptosis of diffuse large B cell lymphoma cells.

C-Myc expression is associated with poor prognosis and aggressive progression of diffuse large B cell lymphoma (DLBCL), and the development of drug-like c-Myc inhibitors remains challenging. In this study, we report a novel berbamine derivative termed 4-chlorobenzoyl berbamine (CBBM) that potently induced the apoptosis of c-Myc-overexpressing DLBCL cells but spared normal blood cells. The compound showed IC50 values ranging from 1.93 to 3.89 µmol/L in DLCBL cells and exhibited a 4.75- to 9.64-fold increase in anti-tumor activity compared to berbamine. Additionally, CBBM inhibited the proliferation of the DLBCL line OCI-Ly3 cells through G0/G1 cell-cycle arrest and induced apoptosis. Further studies have shown that CBBM treatment leads to the proteasome-dependent degradation of c-Myc protein in OCI-Ly3 cells. Interestingly, we found that the inhibitory effect of CBBM was positively correlated with basal levels of CaMKIIγ, which is a key inducer of c-Myc expression in DLBCL cells. We also observed that CBBM inhibits the JAK2/STAT3 pathway, leading to reduced c-Myc transcription. Collectively, these findings suggest that CBBM could be a promising lead compound for treatment of c-Myc-driven DLBCL.

4-Chlorbenzoyl Berbamine, a Novel Derivative of the Natural Product Berbamine, Potently Inhibits the Growth of Human Myeloma Cells by Modulating the NF-κB and JNK Signalling Pathways.

Multiple myeloma (MM) remains incurable despite the development and the use of new agents. In our studies, we found that 4-chlorbenzoyl berbamine (BBMD9), a novel synthetic derivative of berbamine, inhibited the proliferation of MM cells in dose- and time-dependent manners. Flow cytometric (FCM) analysis revealed that MM cells were arrested in the G1 phase and that apoptotic cells increased in a time-dependent manner. Moreover, the BBMD9 treatment downregulated IKKα and IKKß, inhibited p-IκBα, and blocked p65 nuclear localization. Consistently, NF-κB downstream targets, such as cyclinD1 and survivin, were also reduced. In addition, BBMD9 phosphorylated the activity of JNK and c-Jun.

Polyphyllin I induces cell cycle arrest and apoptosis in human myeloma cells via modulating ß-catenin signaling pathway.

Multiple myeloma (MM) is an indolent B-cell disease characterized by clonal proliferation of malignant plasma cells. Multiple myeloma remains incurable despite new targeted drugs and development of drug resistance or intolerable toxicity emerges as a major problem. Therefore, design, identification, and validation of novel chemicals with therapeutic potential are clearly needed for MM treatment. Here, we explore polyphyllin I (PPI), a major active constituent extracted from Paris polyphyllin, its inhibitory effects and its mechanisms in MM cells in vitro. We found that PPI inhibited the proliferation of myeloma cells. The combination of PPI with dexamethasone, doxorubicin, arsenic trioxide, or bortezomib enhanced the inhibition of cell growth. As analyzed by flow cytometry, MM cells were arrested at G2/M phase and apoptotic cells increased in a time-dependent manner. Morphological changes of cells undergoing apoptosis were observed under light microscope. To explore the mechanism of apoptosis induced by PPI, we next examined whether the Wingless-Int (Wnt)/ß-catenin signaling pathway played a role in the PPI-induced growth inhibition in MM cells. The canonical Wnt signaling pathway is activated in MM cells through constitutively active ß-catenin, a messenger molecule relevant to growth, survival, and migration of MM cells. Western blotting was used to measure the protein levels of ß-catenin, and PPI treatment led to downregulating the expression of ß-catenin protein and was followed by inhibition of ß-catenin nuclear localization. As a result, ß-catenin downstream targets, such as cyclin D1 and survivin, were downregulated. To the best of our knowledge, this is the first report identifying anti-proliferative potency of PPI against myeloma cells. PPI blocks ß-catenin nuclear translocation and decreasing expression of the downstream targets of ß-catenin. Our results suggest that PPI is a novel inhibitor of ß-catenin activity with potential anti-myeloma efficacy.

CaMKII γ, a critical regulator of CML stem/progenitor cells, is a target of the natural product berbamine.

Bcr-Abl tyrosine kinase inhibitors (TKIs) have been a remarkable success for the treatment of Ph(+) chronic myeloid leukemia (CML). However, a significant proportion of patients treated with TKIs develop resistance because of leukemia stem cells (LSCs) and T315I mutant Bcr-Abl. Here we describe the unknown activity of the natural product berbamine that efficiently eradicates LSCs and T315I mutant Bcr-Abl clones. Unexpectedly, we identify CaMKII γ as a specific and critical target of berbamine for its antileukemia activity. Berbamine specifically binds to the ATP-binding pocket of CaMKII γ, inhibits its phosphorylation and triggers apoptosis of leukemia cells. More importantly, CaMKII γ is highly activated in LSCs but not in normal hematopoietic stem cells and coactivates LSC-related ß-catenin and Stat3 signaling networks. The identification of CaMKII γ as a specific target of berbamine and as a critical molecular switch regulating multiple LSC-related signaling pathways can explain the unique antileukemia activity of berbamine. These findings also suggest that berbamine may be the first ATP-competitive inhibitor of CaMKII γ, and potentially, can serve as a new type of molecular targeted agent through inhibition of the CaMKII γ activity for treatment of leukemia.

Melanasterasinica He & Burckhardt, sp. nov., a new psylloid species (Hemiptera, Psylloidea, Liviidae) from China developing on Grewia sp. (Malvaceae).

Melanasterasinica He & Burckhardt, sp. nov., a new psylloid species developing on Grewia sp., is described from Hainan, China. It is the first Melanastera species reported from Asia and China, and the second species from the Old World. While New World species of Melanastera are mostly associated with the plant families Melastomataceae and Annonaceae, the two Old World species develop on the malvaceous Grewia, a host otherwise used in psylloids by two Haplaphalara species. The new species is described, diagnosed and illustrated, and its host plant and biogeographic ranges are discussed.

Ectopic expression of the transcription factor ONECUT3 drives a complex karyotype in myelodysplastic syndromes.

Chromosomal instability is a prominent biological feature of myelodysplastic syndromes (MDS), with over 50% of patients with MDS harboring chromosomal abnormalities or a complex karyotype (CK). Despite this observation, the mechanisms underlying mitotic and chromosomal defects in MDS remain elusive. In this study, we identified ectopic expression of the transcription factor ONECUT3, which is associated with CKs and poorer survival outcomes in MDS. ONECUT3-overexpressing cell models exhibited enrichment of several notable pathways, including signatures of sister chromosome exchange separation and mitotic nuclear division with the upregulation of INCENP and CDCA8 genes. Notably, dysregulation of chromosome passenger complex (CPC) accumulation, besides the cell equator and midbody, during mitotic phases consequently caused cytokinesis failure and defective chromosome segregation. Mechanistically, the homeobox (HOX) domain of ONECUT3, serving as the DNA binding domain, occupied the unique genomic regions of INCENP and CDCA8 and transcriptionally activated these 2 genes. We identified a lead compound, C5484617, that functionally targeted the HOX domain of ONECUT3, inhibiting its transcriptional activity on downstream genes, and synergistically resensitized MDS cells to hypomethylating agents. This study revealed that ONECUT3 promoted chromosomal instability by transcriptional activation of INCENP and CDCA8, suggesting potential prognostic and therapeutic roles for targeting high-risk MDS patients with a CK.

An oral triple pill-based cocktail effectively controls acute myeloid leukemia with high translation.

Acute myeloid leukemia (AML) is a deadly hematological malignancy characterized by oncogenic translational addiction that results in over-proliferation and apoptosis evasion of leukemia cells. Various chemo- and targeted therapies aim to reverse this hallmark, but most show only modest efficacy. Here we report a single oral pill containing a low-dose triple small molecule-based cocktail, a highly active anti-cancer therapy (HAACT) with unique mechanisms that can effectively control AML. The cocktail comprises oncogenic translation inhibitor HHT, drug efflux pump P-gpi ENC and anti-apoptotic protein Bcl-2i VEN. Mechanistically, the cocktail can potently kill both leukemia stem cells (LSC) and bulk leukemic cells via co-targeting oncogenic translation, apoptosis machinery, and drug efflux pump, resulting in deep and durable remissions of AML in diverse model systems. We also identified EphB4/Bcl-xL as the cocktail response biomarkers. Collectively, our studies provide proof that a single pill containing a triple combination cocktail might be a promising avenue for AML therapy.

Deletion of IFNγ enhances hepatocarcinogenesis in FXR knockout mice.

BACKGROUND & AIMS: Liver tumor, especially hepatocellular carcinoma (HCC), is closely associated with chronic inflammation. We previously showed that farnesoid X receptor knockout (FXR(-)(/)(-)) mice displayed chronic inflammation and developed spontaneous liver tumors when they aged. However, the mechanism by which inflammation leads to HCC in the absence of FXR is unclear. Because IFNγ is one of the most upregulated pro-inflammatory cytokines in FXR(-)(/)(-) livers, we generated IFNγ(-)(/)(-)FXR(-)(/)(-) double knockout mice to determine IFNγ's roles in hepatocarcinogenesis. METHODS: IFNγ(-)(/)(-) mice were crossed with an FXR(-)(/)(-) C57BL/6 background or injected i.p. with the hepatocarcinogen diethylnitrosamine (DEN). Hepatocarcinogenesis was analyzed with biochemical and histological methods. RESULTS: IFNγ deletion accelerated spontaneous hepatocarcinogenesis in FXR(-)(/)(-) mice and increased the susceptibility to DEN-induced hepatocarcinogenesis. IFNγ deletion enhanced activation of HCC promoters STAT3 and JNK/c-Jun, but abolished induction of p53 in IFNγ(-)(/)(-) livers after acute DEN-induced injury. Furthermore, hepatic p53 expression increased in aged wild type mice but not in aged IFNγ(-)(/)(-) and IFNγ(-)(/)(-)FXR(-)(/)(-) mice, while activation of STAT3 and JNK/c-Jun was enhanced in aged IFNγ(-)(/)(-) and IFNγ(-)(/)(-)FXR(-)(/)(-) mice. In addition, IFNγ inhibited liver cancer xenograft growth and impaired IL-6-induced STAT3 phosphorylation by inducing SOCS1/3 expression. CONCLUSIONS: Increased IFNγ expression in FXR(-)(/)(-) livers represents a protective response of the liver against chronic injury and tumorigenesis. IFNγ suppresses hepatocarcinogenesis by inducing p53 expression and preventing STAT3 activation.

[Hydrogen and Oxygen Isotopic Characteristics of Different Water and Indicative Significance in Baiyangdian Lake].

In order to better understand the water cycle processes in Baiyangdian Lake, samples for precipitation, river water, lake water, and groundwater were collected in 2020 via the analysis of oxygen and hydrogen isotopes and the total dissolved solids. A combined approach including correlation analysis, end-member modeling, and evaporation modeling was used to identify hydrogen and oxygen isotopic characteristics of different water types and their indicative significance to evaporation, hydrodynamics, and lake-groundwater interactions. The results showed that the compositions of hydrogen and oxygen isotopes in surface water and groundwater differed from each other. The average values of δD and δ18O were the highest in samples from lake water in June, secondary for lake water in October, and the lightest in groundwater. The slope of the lake water line was lower than that of the local meteoric water line, which could be attributed to a greater evaporative enrichment in lake water. The water loss ratio was estimated to be 18.8%-42.3% in June and 2.7%-30.3% in October by applying an evaporation model using deuterium excess. Lake water isotopes exhibited highly spatial heterogeneity, which indicated that Baiyangdian Lake was a poor-mixed lake controlled by the complex hydrodynamic conditions. The values of δD and δ18O were lighter around the estuary area and higher in the district far from the estuary. Although the main water isotopes in areas such as channels had a short residence time, the spatial differences in lake water isotopes were not remarkable, which suggested that the well-mixed conditions of the lake water contributed to reducing the heterogeneity of the lake water isotopic compositions on a spatial scale. The contribution ratios of lake leakage to groundwater were 0-91.7% and varied spatially. It had a negative relationship with the buried depth of groundwater and the distance to the lakeshore. No obvious relationship was found between lake leakage magnitude and groundwater level gradient. This study could provide a theoretical basis and technical support for the sustainable development of water resources and ecological environment protection in Baiyangdian Lake.

Aberrantly Activated APOBEC3B Is Associated With Mutant p53-Driven Refractory/Relapsed Diffuse Large B-Cell Lymphoma.

Tumor protein 53 (TP53) mutation predicts an unfavorable prognosis in diffuse large B-cell lymphoma (DLBCL), but the molecular basis for this association remains unclear. In several malignancies, the cytidine deaminase apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) has been reported to be associated with the TP53 G/C-to-A/T mutation. Here, we show that the frequency of this mutation was significantly higher in relapsed/refractory (R/R) than in non-R/R DLBCL, which was positively associated with the APOBEC3B expression level. APOBEC3B overexpression induced the TP53 G/C-to-A/T mutation in vitro, resulting in a phenotype similar to that of DLBCL specimens. Additionally, APOBEC3B-induced p53 mutants promoted the growth of DLBCL cells and enhanced drug resistance. These results suggest that APOBEC3B is a critical factor in mutant p53-driven R/R DLBCL and is therefore a potential therapeutic target.

Identification of Monobenzone as a Novel Potential Anti-Acute Myeloid Leukaemia Agent That Inhibits RNR and Suppresses Tumour Growth in Mouse Xenograft Model.

Acute myeloid leukaemia (AML) is one of the most common types of haematopoietic malignancy. Ribonucleotide reductase (RNR) is a key enzyme required for DNA synthesis and cell proliferation, and its small subunit RRM2 plays a key role for the enzymatic activity. We predicted monobenzone (MB) as a potential RRM2 target compound based on the crystal structure of RRM2. In vitro, MB inhibited recombinant RNR activity (IC50 = 0.25 µM). Microscale thermophoresis indicated that MB inhibited RNR activity by binding to RRM2. MB inhibited cell proliferation (MTT IC50 = 6-18 µM) and caused dose-dependent DNA synthesis inhibition, cell cycle arrest, and apoptosis in AML cells. The cell cycle arrest was reversed by the addition of deoxyribonucleoside triphosphates precursors, suggesting that RNR was the intracellular target of the compound. Moreover, MB overcame drug resistance to the common AML drugs cytarabine and doxorubicin, and treatment with the combination of MB and the Bcl-2 inhibitor ABT-737 exerted a synergistic inhibitory effect. Finally, the nude mice xenografts study indicated that MB administration produced a significant inhibitory effect on AML growth with relatively weak toxicity. Thus, we propose that MB has the potential as a novel anti-AML therapeutic agent in the future.

A Selective Small-Molecule c-Myc Degrader Potently Regresses Lethal c-Myc Overexpressing Tumors.

MYC oncogene is involved in the majority of human cancers and is often associated with poor outcomes, rendering it an extraordinarily desirable target, but therapeutic targeting of c-Myc protein has been a challenge for >30 years. Here, WBC100, a novel oral active molecule glue that selectively degrades c-Myc protein over other proteins and potently kills c-Myc overexpressing cancer cells is reported. WBC100 targets the nuclear localization signal 1 (NLS1)-Basic-nuclear localization signal 2 (NLS2) region of c-Myc and induces c-Myc protein degradation through ubiquitin E3 ligase CHIP mediated 26S proteasome pathway, leading to apoptosis of cancer cells. In vivo, WBC100 potently regresses multiple lethal c-Myc overexpressing tumors such as acute myeloid leukemia, pancreatic, and gastric cancers with good tolerability in multiple xenograft mouse models. Identification of the NLS1-Basic-NLS2 region as a druggable pocket for targeting the "undruggable" c-Myc protein and that single-agent WBC100 potently regresses c-Myc overexpressing tumors through selective c-Myc proteolysis opens new perspectives for pharmacologically intervening c-Myc in human cancers.

Inhibition of the CDK2 and Cyclin A complex leads to autophagic degradation of CDK2 in cancer cells.

Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors against CDK2 are ATP competitors that are either nonspecific or highly toxic, and typically fail clinical trials. One alternative approach is to develop non-ATP competitive inhibitors; they disrupt interactions between CDK2 and either its partners or substrates, resulting in specific inhibition of CDK2 activities. In this report, we identify two potential druggable pockets located in the protein-protein interaction interface (PPI) between CDK2 and Cyclin A. To target the potential druggable pockets, we perform a LIVS in silico screening of a library containing 1925 FDA approved drugs. Using this approach, homoharringtonine (HHT) shows high affinity to the PPI and strongly disrupts the interaction between CDK2 and cyclins. Further, we demonstrate that HHT induces autophagic degradation of the CDK2 protein via tripartite motif 21 (Trim21) in cancer cells, which is confirmed in a leukemia mouse model and in human primary leukemia cells. These results thus identify an autophagic degradation mechanism of CDK2 protein and provide a potential avenue towards treating CDK2-dependent cancers.

miR-194 is a marker of hepatic epithelial cells and suppresses metastasis of liver cancer cells in mice.

UNLABELLED: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by interacting with the 3' untranslated region (3'-UTR) of multiple mRNAs. Recent studies have linked miRNAs to the development of cancer metastasis. In this study, we show that miR-194 is specifically expressed in the human gastrointestinal tract and kidney. Moreover, miR-194 is highly expressed in hepatic epithelial cells, but not in Kupffer cells or hepatic stellate cells, two types of mesenchymal cells in the liver. miR-194 expression was decreased in hepatocytes cultured in vitro, which had undergone a dedifferentiation process. Furthermore, expression of miR-194 was low in liver mesenchymal-like cancer cell lines. The overexpression of miR-194 in liver mesenchymal-like cancer cells reduced the expression of the mesenchymal cell marker N-cadherin and suppressed invasion and migration of the mesenchymal-like cancer cells both in vitro and in vivo. We further demonstrated that miR-194 targeted the 3'-UTRs of several genes that were involved in epithelial-mesenchymal transition and cancer metastasis. CONCLUSION: These results support a role of miR-194, which is specifically expressed in liver parenchymal cells, in preventing liver cancer cell metastasis.

Natural small molecule triptonide inhibits lethal acute myeloid leukemia with FLT3-ITD mutation by targeting Hedgehog/FLT3 signaling.

Acute myeloid leukemia harboring internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD AML) is a subset of highly aggressive malignancies with poor clinical outcome. Despite some advances in the development of FLT3 tyrosine kinase inhibitors (FLT3 inhibitors), most of FLT3-ITD AML patients suffer from lethal disease relapse, suggesting the requirement of novel targets and agents. Here we describe a natural small molecule, triptonide that can efficiently inhibit FLT3-ITD-driven AML in vitro and in vivo. Mechanistically, triptonide targeted Hedgehog/FLT3 signaling by inhibiting its critical effectors, which are GLI2, c-Myc and FLT3 and induced apoptosis of FLT3-ITD-driven leukemia cells. In addition, we also observed that triptonide activated tumor suppressor p53. In vivo, triptonide treatment markedly suppressed lethal FLT3-ITD-driven AML with good tolerance and prolonged survival time in orthotopic mouse model. Our studies identify Hedgehog/FLT3 axis as a novel target for treating FLT3-ITD-driven leukemia and demonstrate that triptonide is an active lead compound that can kill FLT3-ITD-driven leukemia cells.

Unique and complementary suppression of cGAS-STING and RNA sensing- triggered innate immune responses by SARS-CoV-2 proteins.

The emergence of SARS-CoV-2 has resulted in the COVID-19 pandemic, leading to millions of infections and hundreds of thousands of human deaths. The efficient replication and population spread of SARS-CoV-2 indicates an effective evasion of human innate immune responses, although the viral proteins responsible for this immune evasion are not clear. In this study, we identified SARS-CoV-2 structural proteins, accessory proteins, and the main viral protease as potent inhibitors of host innate immune responses of distinct pathways. In particular, the main viral protease was a potent inhibitor of both the RLR and cGAS-STING pathways. Viral accessory protein ORF3a had the unique ability to inhibit STING, but not the RLR response. On the other hand, structural protein N was a unique RLR inhibitor. ORF3a bound STING in a unique fashion and blocked the nuclear accumulation of p65 to inhibit nuclear factor-κB signaling. 3CL of SARS-CoV-2 inhibited K63-ubiquitin modification of STING to disrupt the assembly of the STING functional complex and downstream signaling. Diverse vertebrate STINGs, including those from humans, mice, and chickens, could be inhibited by ORF3a and 3CL of SARS-CoV-2. The existence of more effective innate immune suppressors in pathogenic coronaviruses may allow them to replicate more efficiently in vivo. Since evasion of host innate immune responses is essential for the survival of all viruses, our study provides insights into the design of therapeutic agents against SARS-CoV-2.

The novel human endogenous retrovirus-related gene, psiTPTE22-HERV, is silenced by DNA methylation in cancers.

The psiTPTE22 gene has been designated as a TPTE pseudogene. Our study found that the 5' part of psiTPTE22 has no sequence similarity to TPTE and contains a 3.8-kb human endogenous retrovirus (HERV) element. Because of the HERV element, the 5' part of psiTPTE22 (psiTPTE22-HERV) expresses independently as a gene. Comparison between the DNA sequences of humans and chimps indicated that psiTPTE22-HERV is human specific. We identified 3 alternatively spliced transcript variants from psiTPTE22-HERV by a PCR-based strategy, which use the transcriptional termination signal contained in the HERV element. A 402-nt ORF was contained in the 2 longer transcripts. Western blotting using antibodies produced with chemically synthesized peptide confirmed that a 15-kDa protein was translated from this ORF. RT-PCR results indicated that the ORF-containing transcripts were mainly expressed in psiTPTE22-HERV-expressing samples. Real-time quantitative RT-PCR results showed that expression of the 402-nt ORF was upregulated in normal tissues of kidney, liver, stomach, and lung but downregulated in corresponding tumor tissues. This gene is located near the centromere of chromosome 22 and has a high GC content around the promoter region. Bisulfite sequencing PCR results indicated that it is silenced in cancers by DNA methylation. The expression of psiTPTE22-HERV can be recovered in cancer cells using DNA methylation and histone deacetylase inhibitors. These results suggest psiTPTE22-HERV is regulated epigenetically by DNA methylation. Our study paved the way for further study on an interesting HERV-related human-specific gene, which is silenced in cancers by DNA methylation.

A patch of positively charged amino acids surrounding the human immunodeficiency virus type 1 Vif SLVx4Yx9Y motif influences its interaction with APOBEC3G.

The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K(22)x(3)K(26)x(3)Y(30)x(9)YRHHY(44)) and a cluster of hydrophobic residues (V(55)xIPLx(4-5)LxPhix2YWxL(72)) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.