Comparative characterization of putative chitin deacetylases from Phaeodactylum tricornutum and Thalassiosira pseudonana highlights the potential for distinct chitin-based metabolic processes in diatoms.

Chitin is generally considered to be present in centric diatoms but not in pennate species. Many aspects of chitin biosynthetic pathways have not been explored in diatoms. We retrieved chitin metabolic genes from pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom genomes.

Downregulation of mitochondrial alternative oxidase affects chloroplast function, redox status and stress response in a marine diatom.

Diatom dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria.

PRP-19, a conserved pre-mRNA processing factor and E3 ubiquitin ligase, inhibits the nuclear accumulation of GLP-1/Notch intracellular domain.

The Notch signalling pathway is a conserved and widespread signalling paradigm, and its misregulation has been implicated in numerous disorders, including cancer. The output of Notch signalling depends on the nuclear accumulation of the Notch receptor intracellular domain (ICD). Using the germline, where GLP-1/Notch-mediated signalling is essential for maintaining stem cells, we monitored GLP-1 We found that the nuclear enrichment of GLP-1 ICD is dynamic: while the ICD is enriched in germ cell nuclei during larval development, it is depleted from the nuclei in adult germlines.

The NEDD8 E3 ligase DCNL5 is phosphorylated by IKK alpha during Toll-like receptor activation.

The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue.

BORA-dependent PLK1 regulation: A new weapon for cancer therapy?

The mitotic kinase polo like kinase 1 (PLK1) is overexpressed in many cancers and its inhibition slows down proliferation and increases apoptosis in cancer cell lines. Understanding how PLK1 is activated is therefore crucial for the development of novel PLK1 inhibitors with anticancer properties. We recently identified a conserved regulatory loop leading to PLK1 activation that involves cyclin-dependent kinase 1 (CDK1).

Mitotic entry: The interplay between Cdk1, Plk1 and Bora.

Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase.

Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells.

The conserved Bora protein is a Plk1 activator, essential for checkpoint recovery after DNA damage in human cells. Here, we show that Bora interacts with Cyclin B and is phosphorylated by Cyclin B/Cdk1 at several sites. The first 225 amino acids of Bora, which contain two Cyclin binding sites and three conserved phosphorylated residues, are sufficient to promote Plk1 phosphorylation by Aurora A in vitro.

Characterization of the mammalian family of DCN-type NEDD8 E3 ligases.

Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners.

An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum.

Background: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms.

The COP9 signalosome is vital for timely repair of DNA double-strand breaks.

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target.

Kizuna is a novel mitotic substrate for CDC25B phosphatase.

CDC25 dual-specificity phosphatases play a central role in cell cycle control through the activation of Cyclin-Dependent Kinases (CDKs). Expression during mitosis of a stabilized CDC25B mutant (CDC25B-DDA), which cannot interact with the F-box protein βTrCP for proteasome-dependent degradation, causes mitotic defects and chromosome segregation errors in mammalian cells. We found, using the same CDC25B mutant, that stabilization and failure to degrade CDC25B during mitosis lead to the appearance of multipolar spindle cells resulting from a fragmentation of pericentriolar material (PCM) and abolish mitotic Plk1-dependent phosphorylation of Kizuna (Kiz), which is essential for the function of Kiz in maintaining spindle pole integrity.

A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and facilitates uptake.

Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean [1]. Iron bioavailability has drastically decreased in the contemporary ocean [1], most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton.

Re-print of "Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana".

Post-translational modifications of histones affect many biological processes by influencing higher order chromatin structure that affects gene and genome regulation. It is therefore important to develop methods for extracting histones while maintaining their native post-translational modifications. While histone extraction protocols have been developed in multicellular and single celled organisms such as yeast and Arabidopsis, they are inefficient in diatoms that have a silica cell wall that is likely to hinder histone extraction.

Histone extraction protocol from the two model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana.

Post-translational modifications of histones affect many biological processes by influencing higher order chromatin structure that affects gene and genome regulation. It is therefore important to develop methods for extracting histones while maintaining their native post-translational modifications. While histone extraction protocols have been developed in multicellular and single celled organisms such as yeast and Arabidopsis, they are inefficient in diatoms that have a silica cell wall that is likely to hinder histone extraction.

CSN- and CAND1-dependent remodelling of the budding yeast SCF complex.

Cullin-RING ligases (CRLs) are ubiquitin E3 enzymes with variable substrate-adaptor and -receptor subunits. All CRLs are activated by modification of the cullin subunit with the ubiquitin-like protein Nedd8 (neddylation). The protein CAND1 (Cullin-associated-Nedd8-dissociated-1) also promotes CRL activity, even though it only interacts with inactive ligase complexes.

NEDD8 overexpression results in neddylation of ubiquitin substrates by the ubiquitin pathway.

Ubiquitin and ubiquitin-like proteins use unique E1, E2, and E3 enzymes for conjugation to their substrates. We and others have recently reported that increases in the relative concentration of the ubiquitin-like protein NEDD8 over ubiquitin lead to activation of NEDD8 by the ubiquitin E1 enzyme. We now show that this results in erroneous conjugation of NEDD8 to ubiquitin substrates, such as p53, Caspase 7, and Hif1α, demonstrating that overexpression of NEDD8 is not appropriate for identification of substrates of the NEDD8 pathway.

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes.

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins.

βTrCP-dependent degradation of CDC25B phosphatase at the metaphase-anaphase transition is a pre-requisite for correct mitotic exit.

The dual-specificity phosphatase CDC25B, a key regulator of CDK/Cyclin complexes, is considered as the starter of mitosis. It is an unstable protein, degraded by the proteasome, but often overexpressed in various human cancers. Based on experiments carried out in Xenopus eggs, and on video microscopy studies in mammalian cells, it has been proposed that human CDC25B degradation is dependent of the F-box protein βTrCp, but the involvement of this latter protein was not formally demonstrated yet.

A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking.

In eukaryotic cells, proteasomes play an essential role in intracellular proteolysis and are involved in the control of most biological processes through regulated degradation of key proteins. Analysis of 20S proteasome localization in human cell lines, using ectopic expression of its CFP-tagged alpha7 subunit, revealed the presence in nuclear foci of a specific and proteolytically active complex made by association of the 20S proteasome with its PA28gamma regulator. Identification of these foci as the nuclear speckles (NS), which are dynamic subnuclear structures enriched in splicing factors (including the SR protein family), prompted us to analyze the role(s) of proteasome-PA28gamma complexes in the NS.

The absence of p53 aggravates polyploidy and centrosome number abnormality induced by Aurora-C overexpression.

Aurora-C is the third member of the aurora serine/threonine kinase family and was found only in mammals. Because Aurora-C is overexpressed in many different types of cancer cells we decided to analyze the consequences of Aurora-C overexpression in human cells. We first investigated the subcellular localization of overexpressed GFP-Aurora-C in mitosis and interphase in HeLa cells.