A motor independent requirement for dynein light chain in Caenorhabditis elegans meiotic synapsis.

The dynein motor complex is thought to aid in homolog pairing in many organisms by moving chromosomes within the nuclear periphery to promote and test homologous interactions. This precedes synaptonemal complex (SC) formation during homolog synapsis, which stabilizes homolog proximity during recombination. We observed that depletion of the dynein light chain (DLC-1) in Caenorhabditis elegans irreversibly prevents synapsis, causing an increase in off-chromatin formation of SC protein foci with increasing temperature. This requirement for DLC-1 is independent of its function in dynein motors, as SYP protein foci do not form with depletion of other dynein motor components. In contrast to normal SC-related structures, foci formed with DLC-1 depletion are resistant to dissolution with 1,6-hexanediol, similar to aggregates of SC proteins formed in high growth temperatures. Dynein light chains have been shown to act as hub proteins that interact with other proteins through a conserved binding motif. We identified a similar DLC-1 binding motif in the C. elegans SC protein SYP-2, and mutation of the putative motif causes meiosis defects that are exacerbated by elevated temperatures. We propose that DLC-1 acts as a pre-synapsis chaperone-like factor for SYP proteins to help regulate their self-association prior to the signals for SC assembly, a role that is revealed by its increased essentiality at elevated temperatures.

Discovery of a Highly Potent and Novel Gambogic Acid Derivative as an Anticancer Drug Candidate.

BACKGROUND AND PURPOSE: Gambogic Acid (GA), a promising anti-cancer agent isolated from the resin of Garcinia species in Southeast Asia, exhibits high potency in inhibiting a wide variety of cancer cells' growth. Moreover, the fact that it is amenable to chemical modification makes GA an attractive molecule for the development of anti-cancer agents. METHODS: Gambogic acid-3-(4-pyrimidinyloxy) propyl ester (compound 4) was derived from the reaction between 4-hydroxypropoxy pyrimidine and GA. Its structure was elucidated by comprehensive analysis of ESIMS, HRESIMS, 1 D NMR data. Anti-tumor activities of compound 4 and GA in vitro against HepG-2, A549 and MCF-7 cells were investigated by MTT assay. FITC/PI dye was used to test apoptosis. The binding affinity difference of compound 4 and GA binding to IKKß was studied by using Discovery Studio 2016. RESULTS: Compound 4 was successfully synthesized and showed strong inhibitory effects on HepG-2, A549 and MCF-7 cells lines with an IC50 value of 1.49±0.11, 1.37±0.06 and 0.64±0.16µM, respectively. Molecular docking study demonstrated that four more hydrogen bonds were established between IKKß and compound 4, compared with GA. CONCLUSION: Our results suggested that compound 4 showed significant effects in inducing apoptosis. Further molecular docking study indicated that the introduction of pyrimidine could improve GA's binding affinity to IKKß. Compound 4 may serve as a potential lead compound for the development of new anti-cancer drugs.

MIB-1 Is Required for Spermatogenesis and Facilitates LIN-12 and GLP-1 Activity in Caenorhabditis elegans.

Covalent attachment of ubiquitin to substrate proteins changes their function or marks them for proteolysis, and the specificity of ubiquitin attachment is mediated by the numerous E3 ligases encoded by animals. Mind Bomb is an essential E3 ligase during Notch pathway signaling in insects and vertebrates. While Caenorhabditis elegans encodes a Mind Bomb homolog (mib-1), it has never been recovered in the extensive Notch suppressor/enhancer screens that have identified numerous pathway components. Here, we show that C. elegans mib-1 null mutants have a spermatogenesis-defective phenotype that results in a heterogeneous mixture of arrested spermatocytes, defective spermatids, and motility-impaired spermatozoa. mib-1 mutants also have chromosome segregation defects during meiosis, molecular null mutants are intrinsically temperature-sensitive, and many mib-1 spermatids contain large amounts of tubulin. These phenotypic features are similar to the endogenous RNA intereference (RNAi) mutants, but mib-1 mutants do not affect RNAi. MIB-1 protein is expressed throughout the germ line with peak expression in spermatocytes followed by segregation into the residual body during spermatid formation. C. elegans mib-1 expression, while upregulated during spermatogenesis, also occurs somatically, including in vulva precursor cells. Here, we show that mib-1 mutants suppress both lin-12 and glp-1 (C. elegans Notch) gain-of-function mutants, restoring anchor cell formation and a functional vulva to the former and partly restoring oocyte production to the latter. However, suppressed hermaphrodites are only observed when grown at 25°, and they are self-sterile. This probably explains why mib-1 was not previously recovered as a Notch pathway component in suppressor/enhancer selection experiments.

Grincamycins I - K, Cytotoxic Angucycline Glycosides Derived from Marine-Derived Actinomycete Streptomyces lusitanus SCSIO LR32.

Three new angucycline glycosides, designated grincamycin I (1: ), J (2: ), and K (3: ), together with the known congener A-7884 (4: ), were isolated from marine-derived actinomycete Streptomyces lusitanus SCSIO LR32. The structures of the new compounds were elucidated by comprehensive spectral data analysis. Compounds 2: and 4: exhibited antitumor activity against human cancer cells MDA-MB-435, MDA-MB-231, NCI-H460, HCT-116 and HepG2, and human normal breast epithelial cell MCF10A with IC50 values ranging from 0.4 to 6.9 µM. In addition, A-7884 (4: ) demonstrated antimicrobial activity against Micrococcus luteus with an MIC value of 1.95 µg/mL.

Apicidin Inhibited Proliferation and Invasion and Induced Apoptosis via Mitochondrial Pathway in Non-small Cell Lung Cancer GLC-82 Cells.

BACKGROUND: Apicidin, as an inhibitor of histone deacetylase, showed a wide range of antiproliferative activity against various cancer cell lines. Apicidin has also been reported to induce apotosis via Fas/Fas ligand. Yet few studies have been focused on mitochondrial pathway for its anti-tumor activity. OBJECTIVE: In this study, we evaluated its involved mitochondrial mechanism against non-small cell lung cancer GLC-82 cells. METHOD: Apicidin was isolated from the mangrove endophtic fungi Fusarium sp. by solvent extraction and column chromatography. Its structure was elucidated by MS and NMR spectroscopic data, and comparison of those data with published data. Furthermore, anti-tumor activity and mitochondrial pathway of apicidin against GLC-82 cells were studied. RESULTS: Apicidin was obtained from secondary metabolites of Fusarium sp., and it showed potent inhibitory activity against GLC-82 cells with the IC50 value of 6.94 ± 0.27 µM. Furthermore, apicidin suppressed proliferation and invasion, and induced apoptosis via mitochondrial pathway in GLC-82 cells, including loss of ΔΨm, release of cytochrome c from mitochondria, activation of caspase-9 and -3, and cleavage of poly-ADP-ribose polymerase. CONCLUSION: Apicidin, an inhibitor of histone deacetylase obtained from the mangrove endophytic fungi Fusarium sp., not only inhibited proliferation and invasion of GLC-82 cells, but also induced apoptosis via the mitochondrial pathway.

The Drosophila Helicase Maleless (MLE) is Implicated in Functions Distinct From its Role in Dosage Compensation.

Helicases are ubiquitous enzymes that unwind or remodel single or double-stranded nucleic acids, and that participate in a vast array of metabolic pathways. The ATP-dependent DEXH-box RNA/DNA helicase MLE was first identified as a core member of the chromatin remodeling MSL complex, responsible for dosage compensation in Drosophila males. Although this complex does not assemble in females, MLE is present. Given the multiplicity of functions attributed to its mammalian ortholog RNA helicase A, we have carried out an analysis for the purpose of determining whether MLE displays the same diversity. We have identified a number of different proteins that associate with MLE, implicating its role in specific pathways. We have documented this association in selected examples that include the spliceosome complex, heterogeneous Nuclear Ribonucleoproteins involved in RNA Processing and in Heterochromatin Protein 1 deposition, and the NuRD complex.

Topoisomerase II plays a role in dosage compensation in Drosophila.

In Drosophila, dosage compensation is mediated by the MSL complex, which binds numerous sites on the X chromosome in males and enhances the transcriptional rate of a substantial number of X-linked genes. We have determined that topoisomerase II (Topo II) is enriched on dosage compensated genes, to which it is recruited by association with the MSL complex, in excess of the amount that is present on autosomal genes with similar transcription levels. Using a plasmid model, we show that Topo II is required for proper dosage compensation and that compensated chromatin is topologically different from non-compensated chromatin. This difference, which is not the result of the enhanced transcription level due of X-linked genes and which represents a structural modification intrinsic to the DNA of compensated chromatin, requires the function of Topo II. Our results suggest that Topo II is an integral part of the mechanistic basis of dosage compensation.

Distinct contributions of MSL complex subunits to the transcriptional enhancement responsible for dosage compensation in Drosophila.

The regulatory mechanism of dosage compensation is the paramount example of epigenetic regulation at the chromosomal level. In Drosophila, this mechanism, designed to compensate for the difference in the dosage of X-linked genes between the sexes, depends on the MSL complex that enhances the transcription of the single dose of these genes in males. We have investigated the function of various subunits of the complex in mediating dosage compensation. Our results confirm that the highly enriched specific acetylation of histone H4 at lysine 16 of compensated genes by the histone acetyl transferase subunit MOF induces a more disorganized state of their chromatin. We have determined that the association of the MSL complex reduces the level of negative supercoiling of the deoxyribonucleic acid of compensated genes, and we have defined the role that the other subunits of the complex play in this topological modification. Lastly, we have analyzed the potential contribution of ISWI-containing remodeling complexes to the architecture of compensated chromatin, and we suggest a role for this remodeling factor in dosage compensation.

Role of the ATPase/helicase maleless (MLE) in the assembly, targeting, spreading and function of the male-specific lethal (MSL) complex of Drosophila.

BACKGROUND: The male-specific lethal (MSL) complex of Drosophila remodels the chromatin of the X chromosome in males to enhance the level of transcription of most X-linked genes, and thereby achieve dosage compensation. The core complex consists of five proteins and one of two non-coding RNAs. One of the proteins, MOF (males absent on the first), is a histone acetyltransferase that specifically acetylates histone H4 at lysine 16. Another protein, maleless (MLE), is an ATP-dependent helicase with the ability to unwind DNA/RNA or RNA/RNA substrates in vitro. Recently, we showed that the ATPase activity of MLE is sufficient for the hypertranscription of genes adjacent to a high-affinity site by MSL complexes located at that site. The helicase activity is required for the spreading of the complex to the hundreds of positions along the X chromosome, where it is normally found. In this study, to further understand the role of MLE in the function of the MSL complex, we analyzed its relationship to the other complex components by creating a series of deletions or mutations in its putative functional domains, and testing their effect on the distribution and function of the complex in vivo. RESULTS: The presence of the RB2 RNA-binding domain is necessary for the association of the MSL3 protein with the other complex subunits. In its absence, the activity of the MOF subunit was compromised, and the complex failed to acetylate histone H4 at lysine 16. Deletion of the RB1 RNA-binding domain resulted in complexes that maintained substantial acetylation activity but failed to spread beyond the high-affinity sites. Flies bearing this mutation exhibited low levels of roX RNAs, indicating that these RNAs failed to associate with the proteins of the complex and were degraded, or that MLE contributes to their synthesis. Deletion of the glycine-rich C-terminal region, which contains a nuclear localization sequence, caused a substantial level of retention of the other MSL proteins in the cytoplasm. These data suggest that the MSL proteins assemble into complexes or subcomplexes before entering the nucleus. CONCLUSIONS: This study provides insights into the role that MLE plays in the function of the MSL complex through its association with roX RNAs and the other MSL subunits, and suggests a hypothesis to explain the role of MLE in the synthesis of these RNAs.

A plasmid model system shows that Drosophila dosage compensation depends on the global acetylation of histone H4 at lysine 16 and is not affected by depletion of common transcription elongation chromatin marks.

Dosage compensation refers to the equalization of most X-linked gene products between males, which have one X chromosome and a single dose of X-linked genes, and females, which have two X's and two doses of such genes. We developed a plasmid-based model of dosage compensation that allows new experimental approaches for the study of this regulatory mechanism. In Drosophila melanogaster, an enhanced rate of transcription of the X chromosome in males is dependent upon the presence of histone H4 acetylated at lysine 16. This chromatin mark occurs throughout active transcriptional units, leading us to the conclusion that the enhanced level of transcription is achieved through an enhanced rate of RNA polymerase elongation. We used the plasmid model to demonstrate that enhancement in the level of transcription does not depend on other histone marks and factors that have been associated with the process of elongation, thereby highlighting the special role played by histone H4 acetylated at lysine 16 in this process.