Development and evaluation of 4-(pyrrolidin-3-yl)benzonitrile derivatives as inhibitors of lysine specific demethylase 1

As part of our ongoing efforts to develop reversible inhibitors of LSD1, we identified a series of 4-(pyrrolidin-3-yl)benzonitrile derivatives that act as successful scaffold-hops of the literature inhibitor GSK-690. The most active compound, 21g, demonstrated a K_d value of 22 nM and a biochemical IC₅₀ of 57 nM. In addition, this compound displayed improved selectivity over the hERG ion channel compared to GSK-690, and no activity against the related enzymes MAO-A and B. In human THP-1 acute myeloid leukaemia cells, 21g was found to increase the expression of the surrogate cellular biomarker CD86. This work further demonstrates the versatility of scaffold-hopping as a method to develop structurally diverse, potent inhibitors of LSD1.     

COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

Mobilizable carbon reserves in young peach trees as evidenced by trunk girdling experiments

The seasonal patterns in concentrations of both soluble (NSC-S)and insoluble (NSC-I) non-structural carbohydrates, in 3-year-oldpeach trees (Prunus persica L. Batsch) grown in sand cultureare described. The ability of trees to mobilize their carbohydratereserves in response to scion-trunk girdling, which preventsphotosynthate transport toward the roots, was tested at fourphenological stages. Girdling induces a NSC-I depletion in rootsand rootstock-trunk bark and a NSC-I accumulation in leavesand shoots. On the contrary, the NSC-S concentrations of theorgans located both above and below girdling were not significantlyaffected by the treatment. Consequently, when phloem transportbreaks down, trees, whatever their growing stage, mobilize carbohydratereserves below the girdle to maintain the soluble sugar contentsat the same level as in control trees. Key words: Girdling, non-structural carbohydrates, Prunus persica L., carbon reserves, seasonal patterns     

Sequence and secondary structure of Drosophila melanogaster 5.8S and 2S rRNAs and of the processing site between them.

Drosophila melanogaster 5.8S and 2S rRNAs were end-labeled with 32p at either the 5' or 3' end and were sequenced. 5.8S rRNA is 123 nucleotides long and homologous to the 5' part of sequenced 5.8S molecules from other species. 2S rRNA is 30 nucleotides long and homologous to the 3' part of other 5.8S molecules. The 3' end of the 5.8S molecule is able to base-pair with the 5' end of the 2S rRNA to generate a helical region equivalent in position to the "GC-rich hairpin" found in all previously sequenced 5.8S molecules. Probing the structure of the labeled Drosophila 5.8S molecule with S1 nuclease in solution verifies its similarity to other 5.8S rRNAs. The 2S rRNA is shown to form a stable complex with both 5.8S and 26S rRNAs separately and together. 5.8S rRNA can also form either binary or ternary complexes with 2S and 26S rRNA. It is concluded that the 5.8S rRNA in Drosophila melanogaster is very similar both in sequence and structure to other 5.8 rRNAs but is split into two pieces, the 2S rRNA being the 3' part. 2S anchors the 5.8S and 26S rRNA. The order of the rRNA coding regions in the ribosomal DNA repeating unit is shown to be 18S - 5.8S - 2S - 26S. Direct sequencing of ribosomal DNA shows that the 5.8S and 2S regions are separated by a 28 nucleotide spacer which is A-T rich and is presumably removed by a specific processing event. A secondary structure model is proposed for the 26S-5.8S ternary complex and for the presumptive precursor molecule.     

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.     

Complete nucleotide sequence of a gene encoding a functional human class I histocompatibility antigen (HLA-CW3)

The HLA-CW3 gene contained in a cosmid clone identified by transfection expression experiments has been completely sequenced. This provides, for the first time, data on the structure of HLA-C locus products and constitutes, together with that of the gene coding for HLA-A3, the first complete nucleotide sequences of genes coding for serologically defined class I HLA molecules. In contrast to the organisation of the two class I HLA pseudogenes whose sequences have previously been determined, the sequence of the HLA-CW3 gene reveals an additional cytoplasmic encoding domain, making the organisation of this gene very similar to that of known H-2 class I genes and also the HLA-A3 gene. The deduced amino acid sequences of HLA-CW3 and HLA-A3 now allow a systematic comparison of such sequences of HLA class I molecules from the three classical transplantation antigen loci A, B, C. The compared sequences include the previously determined partial amino acid sequences of HLA-B7, HLA-B40, HLA-A2 and HLA-A28. The comparisons confirm the extreme polymorphism of HLA classical class I molecules, and permit a study of the level of diversity and the location of sequence differences. The distribution of differences is not uniform, most of them being located in the first and second extracellular domains, the third extracellular domain is extremely conserved, and the cytoplasmic domain is also a variable region. Although it is difficult to determine locus-specific regions, we have identified several candidate positions which may be C locus-specific.     

Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence

In this study, the role of Listeria monocytogenes ferritin was investigated. The fri gene encoding the ferritin was deleted and the phenotype of the mutant was analyzed demonstrating that ferritin is necessary for optimal growth in minimal medium in both presence and absence of iron, as well as after cold- and heat-shock. We also showed that ferritin provides protection against reactive oxygen species and is essential for full virulence of L. monocytogenes. A comparative proteomic analysis revealed an effect of the fri deletion on the levels of listeriolysin O and several stress proteins. Together, our study demonstrates that fri has multiple roles that contribute to Listeria virulence.