Analysis of Wild Type LbCpf1 Protein, and PAM Recognition Variants, in a Cellular Context.

Nucleases used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. So far CRISPR-Cas, the RNA-guided nucleases, is the most advanced genome engineering system. The CRISPR nucleases allows recognition of a particular genomic sequence with two distinct molecular interactions: first, by direct interaction between the nuclease and the protospacer-adjacent motif, wherein discrete amino acids interact with DNA base pairs; and second, by hybridization of the guide RNA with the target DNA sequence.

Dimerization, oligomerization, and aggregation of human amyotrophic lateral sclerosis copper/zinc superoxide dismutase 1 protein mutant forms in live cells.

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol.

Identification of a novel sulfonamide non-nucleoside reverse transcriptase inhibitor by a phenotypic HIV-1 full replication assay.

Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated.

Generation and functional analysis of zinc finger nucleases.

The recent development of artificial endonucleases with tailored specificities has opened the door for a wide range of new applications, including the correction of mutated genes directly in the chromosome. This kind of gene therapy is based on homologous recombination, which can be stimulated by the creation of a targeted DNA double-strand break (DSB) near the site of the desired recombination event. Artificial nucleases containing zinc finger DNA-binding domains have provided important proofs of concept, showing that inserting a DSB in the target locus leads to gene correction frequencies of 1-18% in human cells.

The CD44 standard/ezrin complex regulates Fas-mediated apoptosis in Jurkat cells.

The transmembrane receptor CD44 conveys important signals from the extracellular microenvironment to the cytoplasm, a phenomena known as "outside-in" signaling. CD44 exists as several isoforms that result from alternative splicing, which differ only in the extracellular domain but yet exhibit different activities. CD44 is a binding partner for the membrane-cytoskeleton cross-linker protein ezrin.

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases.

Artificial endonucleases consisting of a FokI cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off-target sites.

Discovery of novel, highly potent and selective beta-hairpin mimetic CXCR4 inhibitors with excellent anti-HIV activity and pharmacokinetic profiles.

Novel highly potent CXCR4 inhibitors with good pharmacokinetic properties were designed and optimized starting from the naturally occurring beta-hairpin peptide polyphemusin II. The design involved incorporating important residues from polyphemusin II into a macrocyclic template-bound beta-hairpin mimetic. Using a parallel synthesis approach, the potency and ADME properties of the mimetics were optimized in iterative cycles, resulting in the CXCR4 inhibitors POL2438 and POL3026.

The peptidyl-prolyl isomerase Pin1 regulates phospho-Ser77 retinoic acid receptor alpha stability.

Peptidyl-prolyl isomerases (PPIase) facilitate the cis-trans interconversion of the peptidyl-prolyl bond and in such way affect protein folding. Pin1 is a PPIase, which specifically recognizes phosphorylated S/T-P bonds. The transcription factor TFIIH mediates phosphorylation of the retinoic acid receptor alpha (RARalpha) at position Ser77.

Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A.

Human immunodeficiency virus 1 (HIV-1) multiplication depends on a cellular protein, cyclophilin A (CyPA), that gets integrated into viral particles. Because CyPA is not required for cell viability, we attempted to block its synthesis in order to inhibit HIV-1 replication. For this purpose, we used antisense U7 small nuclear RNAs (snRNAs) that disturb CyPA pre-mRNA splicing and short interfering RNAs (siRNAs) that target CyPA mRNA for degradation.

Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP.

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer.

Promoter of FGF8 reveals a unique regulation by unliganded RARalpha.

We previously reported that retinoids were inducing a complete switch in the expression of two isoforms from the fgf8 gene. In order to gain insight into the transcriptional mechanisms possibly involved in this regulation, we cloned and sequenced a fragment of genomic DNA encompassing 6 kb of the region 5' upstream of the fgf8 coding sequence and investigated its promoter elements. A comprehensive series of biochemical and cellular experiments determined two distinct functional regions cis-responsive to retinoids and/or their receptors: (i) a canonical RARE (type DR2) which is the cis target of a RARalpha-RXRalpha liganded heterodimer; and (ii) a completely novel type of response element composed of two half-binding sites separated by 87 nucleotides, which we demonstrate to be the target of an unliganded RARalpha homodimer phosphorylated on the Ser77 residue.