Differential properties of transcriptional complexes formed by the CoREST family

Mammalian genomes harbor three CoREST genes. rcor1 encodes CoREST (CoREST1) and the paralogues rcor2 and rcor3 encode CoREST2 and CoREST3, respectively. Here, we describe specific properties of transcriptional complexes formed by CoREST proteins with the histone demethylase LSD1/KDM1A and histone deacetylases HDAC1/2 and the finding that all three CoRESTs express in the adult rat brain. CoRESTs interact equally strong with LSD1/KDM1A. Structural analysis shows that the overall conformation of CoREST3 is similar to that of CoREST1 complexed with LSD1/KDM1A. Nonetheless, transcriptional repressive capacity of CoREST3 is lower than that of CoREST1, which correlates with the observation that CoREST3 leads to a reduced LSD1/KDM1A catalytic efficiency. Also, CoREST2 shows a lower transcriptional repression than CoREST1, which is resistant to HDAC inhibitors. CoREST2 displays lower interaction with HDAC1/2 which is barely present in LSD1/KDM1A-CoREST2 complexes. A non-conserved Leucine in the first SANT domain of CoREST2 severely weakens its association to HDAC1/2. Furthermore, CoREST2 mutants with either increased or lacking HDAC1/2 interaction feature equivalent transcriptional repression capacities, indicating that CoREST2 represses in a HDAC-independent manner. In conclusion, differences among CoREST proteins are instrumental to the modulation of protein-protein interactions and catalytic activities of LSD1/KDM1A-CoREST-HDAC complexes, fine tuning gene expression regulation

Protein Recognition by Short Peptide Reversible Inhibitors of the Chromatin-Modifying LSD1/CoREST Lysine Demethylase.

The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1

Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors

A tetracationic porphyrin with dual anti-prion activity

Prions are deadly infectious agents made of PrPSc, a misfolded variant of the cellular prion protein (PrPC) which self-propagates by inducing misfolding of native PrPC. PrPSc can adopt different pathogenic conformations (prion strains), which can be resistant to potential drugs, or acquire drug resistance, hampering the development of effective therapies. We identified Zn(II)-BnPyP, a tetracationic porphyrin that binds to distinct domains of native PrPC, eliciting a dual anti-prion effect. Zn(II)-BnPyP binding to a C-terminal pocket destabilizes the native PrPC fold, hindering conversion to PrPSc; Zn(II)-BnPyP binding to the flexible N-terminal tail disrupts N- to C-terminal interactions, triggering PrPC endocytosis and lysosomal degradation, thus reducing the substrate for PrPSc generation. Zn(II)-BnPyP inhibits propagation of different prion strains in vitro, in neuronal cells and organotypic brain cultures. These results identify a PrPC-targeting compound with an unprecedented dual mechanism of action which might be exploited to achieve anti-prion effects without engendering drug resistance

Differential properties of transcriptional complexes formed by the CoREST family.

Mammalian genomes harbor three CoREST genes. rcor1 encodes CoREST (CoREST1) and the paralogues rcor2 and rcor3 encode CoREST2 and CoREST3, respectively. Here, we describe specific properties of transcriptional complexes formed by CoREST proteins with the histone demethylase LSD1/KDM1A and histone deacetylases HDAC1/2 and the finding that all three CoRESTs express in the adult rat brain. CoRESTs interact equally strong with LSD1/KDM1A. Structural analysis shows that the overall conformation of CoREST3 is similar to that of CoREST1 complexed with LSD1/KDM1A. Nonetheless, transcriptional repressive capacity of CoREST3 is lower than that of CoREST1, which correlates with the observation that CoREST3 leads to a reduced LSD1/KDM1A catalytic efficiency. Also, CoREST2 shows a lower transcriptional repression than CoREST1, which is resistant to HDAC inhibitors. CoREST2 displays lower interaction with HDAC1/2 which is barely present in LSD1/KDM1A-CoREST2 complexes. A non-conserved Leucine in the first SANT domain of CoREST2 severely weakens its association to HDAC1/2. Furthermore, CoREST2 mutants with either increased or lacking HDAC1/2 interaction feature equivalent transcriptional repression capacities, indicating that CoREST2 represses in a HDAC-independent manner. In conclusion, differences among CoREST proteins are instrumental to the modulation of protein-protein interactions and catalytic activities of LSD1/KDM1A-CoREST-HDAC complexes, fine tuning gene expression regulation

Structural and biochemical evaluation of Ceratitis capitata odorant-binding protein 22 affinity for odorants involved in intersex communication

In insects, odorant-binding proteins (OBPs) connect the peripheral sensory system to receptors of olfactory organs. Medfly Ceratitis capitata CcapObp22 shows 37% identity and close phylogenetic affinities with Drosophila melanogaster OBP69a/pheromonebinding protein related protein 1. The CcapObp22 gene is transcribed in the antennae and maxillary palps, suggesting an active role in olfaction. Here, we recombinantly produced CcapObp22, obtaining a 13.5 kDa protein capable of binding multiple strongly hydrophobic terpene compounds, including medfly male pheromone components. The highest binding affinity [half maximal effective concentration (EC50) = 0.48 µM] was to (E,E)-α-farnesene, one of the most abundant compounds in the male pheromone blend. This odorant was used in cocrystallization experiments, yielding the structure of CcapOBP22. The monomeric structure shows the typical OBP folding, constituted by six α-helical elements interconnected by three disulphide bridges. A C-terminal seventh α-helix constitutes the wall of a deep, L-shaped hydrophobic cavity. Analysis of the electron density in this cavity suggested trapping of farnesene in the crystal structure, although with partial occupancy. Superposition of the CcapOBP22 structure with related seven-helical OBPs highlights striking similarity in the organization of the C-terminal segment of these proteins. Collectively, our molecular and physiological data on medfly CcapOBP22 suggest its involvement in intersex olfactory communication

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein-dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl-binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores