Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.     

Inhibitors of histone deacetylase as antitumor agents: A critical review

Histone deacetylase (EC 3.5.1.98 – HDAC) is an amidohydrolase involved in deacetylating the histone lysine residues for chromatin remodeling and thus plays a vital role in the epigenetic regulation of gene expression. Due to its aberrant activity and over expression in several forms of cancer, HDAC is considered as a potential anticancer drug target. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as cell survival, differentiation and apoptosis in tumor cells and thus exhibit anticancer activity. Till date, four drugs, namely Vorinostat (SAHA), Romidepsin (FK-228), Belinostat (PXD-101) and Panobinostat (LBH-589) have been granted FDA approval for cancer and several HDAC inhibitors are currently in various phases of clinical trials, either as monotherapy and/or in combination with existing/novel anticancer agents. Regardless of this, today scientific efforts have fortified the quest for newer and novel HDAC inhibitors that show isoform selectivity. This review focuses on the chemistry of the molecules of two classes of HDAC inhibitors, namely short chain fatty acids and hydroxamic acids, investigated so far as novel therapeutic agents for cancer.     

组蛋白去乙酰化酶抑制剂诱导肿瘤细胞凋亡的机制

组蛋白去乙酰化酶抑制剂（HDACi）是一类新的化疗药物,能够有效抑制组蛋白去乙酰化酶的活性,促进组蛋白及非组蛋白的乙酰化修饰,在转录和翻译后修饰水平调控肿瘤靶蛋白及凋亡相关蛋白的表达和降解,活化凋亡信号通路,诱导肿瘤细胞凋亡。HDACi抑制抗氧化蛋白的表达,提高细胞内活性氧的水平,引起细胞的氧化损伤。因此,氧化损伤诱导的细胞凋亡也是HDACi杀伤肿瘤细胞的重要机制。HDACi诱导细胞凋亡机制的发现将进一步促进HDACi在临床治疗中的应用。     

Effectiveness of the Histone Deacetylase Inhibitor (S)-2 against LNCaP and PC3 Human Prostate Cancer Cells

Histone deacetylase inhibitors (HDACi) represent a promising class of epigenetic agents with anticancer properties. Here, we report that (S)-2, a novel hydroxamate-based HDACi, shown previously to be effective against acute myeloid leukemia cells, was also a potent inducer of apoptosis/differentiation in human prostate LNCaP and PC3 cancer cells. In LNCaP cells (S)-2 was capable of triggering H3/H4 histone acetylation, H2AX phosphorylation as a marker of DNA damage and producing G₀/G₁ cell cycle arrest. Consistently, (S)-2 led to enhanced expression of both the protein and mRNA p21 levels in LNCaP cells but, contrary to SAHA, not in normal non-tumorigenic prostate PNT1A cells. Mechanistic studies demonstrated that (S)-2-induced apoptosis in LNCaP cells developed through the cleavage of pro-caspase 9 and 3 and of poly(ADP-ribose)-polymerase accompanied by the dose-dependent loss of mitochondrial membrane potential. Indeed, the addition of the pan-caspase inhibitor Z-VAD-fmk greatly reduced drug-mediated apoptosis while the antioxidant N-acetyl-cysteine was virtually ineffective. Importantly, preliminary data with nude mice xenografted with LNCaP cells showed that (S)-2 prompted a decrease in the tumor volume and an increase in H2AX phosphorylation within the cancer cells. Moreover, the highly metastatic prostate cancer PC3 cells were also sensitive to (S)-2 that: i) induced growth arrest and moderate apoptosis; ii) steered cells towards differentiation and neutral lipid accumulation; iii) reduced cell invasiveness potential by decreasing the amount of MMP-9 activity and up-regulating TIMP-1 expression; and iv) inhibited cell motility and migration through the Matrigel. Overall, (S)-2 has proven to be a powerful HDACi capable of inducing growth arrest, cell death and/or differentiation of LNCaP and PC3 prostate cancer cells and, due to its low toxicity and efficacy in vivo, might also be of clinical interest to support conventional prostate cancer therapy.     

RNA-binding protein AUF1 represses Dicer expression

MicroRNA (miRNA) biogenesis is tightly regulated by numerous proteins. Among them, Dicer is required for the processing of the precursor (pre-)miRNAs into the mature miRNA. Despite its critical function, the mechanisms that regulate Dicer expression are not well understood. Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3′-untranslated region (UTR). Through these interactions, AUF1 lowered DICER1 mRNA stability, since silencing AUF1 lengthened DICER1 mRNA half-life and increased Dicer expression, while overexpressing AUF1 lowered DICER1 mRNA and Dicer protein levels. Given that Dicer is necessary for the synthesis of mature miRNAs, the lowering of Dicer levels by AUF1 diminished the levels of miRNAs tested, but not the levels of the corresponding pre-miRNAs. In summary, AUF1 suppresses miRNA production by reducing Dicer production.     

A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia

We have previously shown that a snoRNA-derived microRNA, miR2, in Giardia lamblia potentially regulates the expression of 22 variant surface protein (VSP) genes. Here, we identified another miRNA, miR4, also capable of regulating the expression of several VSPs but derived from an unannotated open reading frame (ORF) rather than a snoRNA, suggesting a canonical miRNA biogenesis pathway in Giardia. miR4 represses expression of a reporter containing two miR4 antisense sequences at the 3' UTR without causing a corresponding decrease in the mRNA level. This repression requires the presence of the Giardia Argonaute protein (GlAgo) and is reversed by 2' O-methylated antisense oligo to miR4, suggesting an RNA-induced silencing complex (RISC)-mediated mechanism. Furthermore, in vivo and in vitro evidence suggested that the Giardia Dicer protein (GlDcr) is required for miR4 biogenesis. Coimmunoprecipitation of miR4 with GlAgo further verified miR4 as a miRNA. A total of 361 potential target sites for miR4 were bioinformatically identified in Giardia, out of which 69 (32.7%) were associated with VSP genes. miR4 reduces the expression of a reporter containing two copies of the target site from VSP (GL50803_36493) at the 3' UTR. Sixteen of the 69 VSP genes were further found to contain partially overlapping miR2 and miR4 targeting sites. Expression of a reporter carrying the two overlapping sites was inhibited by either miR2 or miR4, but the inhibition was neither synergistic nor additive, suggesting a complex mechanism of miRNA regulation of VSP expression and the presence of a rich miRNAome in Giardia.     

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA‐generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain‐derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca²⁺‐dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre‐miR16, was impaired. Decreased production of miR‐16‐5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane‐targeted TRBP. Moreover, miR‐16‐5p or membrane‐targeted TRBP expression blocked BDNF‐induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity‐dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.