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. Here we report the application of the single strand annealing cellular assay to analyze and quantify nuclease activity of wild type and mutant CRISPR-Cpf1. Using this heterologous marker system based on GFP activity, we observed a comparable PAM recognition selectivity with the NGS analysis. The heterologous marker system has revealed that LbCpf1 is a more specific nuclease than AsCpf1 in a cellular context. We controlled the in vitro activity of the Cpf1 nuclease complexes expressed in mammalian cells and demonstrated that they are responsible of the DNA cleavage at the target site. In addition, we generated and tested LbCpf1 variants with several combinations of mutations at the PAM-recognition positions G532, K538 and Y542. Finally, we showed that the results of the in vitro DNA cleavage assay with the wild type and mutants LbCpf1 corroborate with the selection of 6TG resistant cells associated to the genomic disruption of hprt gene.

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. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.

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. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.

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. In this paper, we describe how zinc finger nucleases are assembled by polymerase chain reaction (PCR) and present two methods to assess these custom nucleases quickly in vitro and in a cell-based recombination assay.

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. In this study, we demonstrate that only CD44 standard (CD44s) colocalizes and interacts with the actin cross-linkers ezrin and moesin using well-characterized cell lines engineered to express different CD44 isoforms. Importantly, we also show that the association CD44s-ezrin-actin is an important modulator of Fas-mediated apoptosis. The results highlight a mechanism by which signals from the extracellular milieu regulate intracellular signaling activities involved in programmed cell death.

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. Here we describe a structure-based approach to reducing off-target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.

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 inhibitory potencies of these compounds were confirmed in a series of HIV-1 invasion assays in vitro. POL3026 showed excellent plasma stability, high selectivity for CXCR4, favorable pharmacokinetic properties in the dog, and thus has the potential to become a therapeutic compound for application in the treatment of HIV infections (as an entry inhibitor), cancer (for angiogenesis suppression and inhibition of metastasis), inflammation, and in stem cell transplant therapy.

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. In the presence of retinoic acid ligand (RA), the Ser77 non-phosphorylated receptor is suggested to undergo degradation through the proteasome pathway. Here we provide evidence that Pin1 is able to selectively destabilize RARalpha in a ligand independent-manner. We show that this is caused by RARalpha ubiquitination, which in turn is phosphorylation dependent. The single mutation Ser77>A completely abolishes RARalpha degradation whereas the mutation Ser77>E rescues this effect. In addition, we correlate RARalpha stability to Ser77 phosphorylation required for the ligand independent transcriptional activity on fgf8 promoter. Finally, we show that the ligand-independent Ser77 phosphorylation requires the genuine ligand-binding domain.

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. With dual-specificity U7 snRNAs targeting the 3' and 5' splice sites of CyPA exons 3 or 4, we obtained an efficient skipping of these exons and a strong reduction of CyPA protein. Furthermore, short interfering RNAs targeting two segments of the CyPA coding region strongly reduced CyPA mRNA and protein levels. Upon lentiviral vector-mediated transduction, prolonged antisense effects were obtained for both types of antisense RNAs in the human T-cell line CEM-SS. These transduced CEM-SS cells showed a delayed, and for the siRNAs also reduced, HIV-1 multiplication. Since the two types of antisense RNAs function by different mechanisms, combining the two approaches may result in a synergistic effect.

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. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.

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. Combined activities of these cis and trans-acting factors support a model of a complex regulation of fgf8 expression: by alternative binding to two distinct promoter elements, phosphorylated-unliganded-RARalpha homodimer or its liganded form have two distinct and mutually exclusive trans-activating activities, explaining the expression of two different isoforms of fgf8.