A light-gated potassium channel for sustained neuronal inhibition.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Peptide-mediated delivery of nucleic acids into mammalian cells.

Control of gene expression using RNA interference (RNAi) technology constitutes a method of choice for investigating gene function in mammalian cells. However, like most oligonucleotide-based strategies, the major limitation of interfering RNA is their poor cellular uptake due to low permeability of the cell membrane to nucleic acids. Several strategies have been developed to improve delivery of oligonucleotides both in cultured cells and in vivo. So far, there is no universal method for their delivery, as they all present several limitations. Peptide-based strategies have been demonstrated to improve the cellular uptake of nucleic acids both in cultured cell and in vivo. This chapter describes a new peptide-based gene delivery system, MPG, which forms stable noncovalent complexes with oligonucleotides and promotes their delivery into a large panel of cell lines without the need for prior chemical covalent coupling. Protocols are described for both adherent and suspension cell lines.

Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells.

The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.

SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence.

Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing has been observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover an essential role of the human SIRT6 enzyme in pericentric transcriptional silencing, and we show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, residue K18 of histone H3 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, depletion of these transcripts through RNA interference rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and regulation of acetylated H3K18 at heterochromatin, and demonstrate the pathogenic role of deregulated pericentric transcription in aging- and cancer-related cellular dysfunction.

Proteomic analysis of the SIRT6 interactome: novel links to genome maintenance and cellular stress signaling.

The chromatin regulatory factor SIRT6 plays pivotal roles in metabolism, tumor suppression, and aging biology. Despite the fundamental roles of SIRT6 in physiology and disease, only a handful of molecular and functional interactions of SIRT6 have been reported. Here, we characterize the SIRT6 interactome and identify 80+ novel SIRT6-interacting proteins. The discovery of these SIRT6-associations considerably expands knowledge of the SIRT6 interaction network, and suggests previously unknown functional interactions of SIRT6 in fundamental cellular processes. These include chromatin remodeling, mitotic chromosome segregation, protein homeostasis, and transcriptional elongation. Extended analysis of the SIRT6 interaction with G3BP1, a master stress response factor, uncovers an unexpected role and mechanism of SIRT6 in regulating stress granule assembly and cellular stress resistance.

Biochemical characterization and crystal structure of a Dim1 family associated protein: Dim2.

The U4/U6*U5 tri-snRNP complex is the catalytic core of the pre-mRNA splicing machinery. The thioredoxin-like protein hDim1 (U5-15 kDa) constitutes an essential component of the U5 particle, and its functions have been reported to be highly conserved throughout evolution. Recently, the Dim1-like protein (DLP) family has been extended to other proteins harboring similar sequence motifs. Here we report the biochemical characterization and crystallographic structure of a 149 amino acid protein, hDim2, which shares 38% sequence identity with hDim1. The crystallographic structure of hDim2 solved at 2.5 A reveals a classical thioredoxin-fold structure. However, despite the similarity in the thioredoxin fold, hDim2 differs from hDim1 in many significant features. The structure of hDim2 contains an extra alpha helix (alpha3) and a beta strand (beta5), which stabilize the protein, suggesting that they may be involved in interactions with hDim2-specific partners. The stability and thermodynamic parameters of hDim2 were evaluated by combining circular dichroism and fluorescence spectroscopy together with chromatographic and cross-linking approaches. We have demonstrated that, in contrast to hDim1, hDim2 forms stable homodimers. The dimer interface is essentially stabilized by electrostatic interactions and involves tyrosine residues located in the alpha3 helix. Structural analysis reveals that hDim2 lacks some of the essential structural motifs and residues that are required for the biological activity and interactive properties of hDim1. Therefore, on the basis of structural investigations we suggest that, in higher eukaryotes, although both hDim1 and hDim2 are involved in pre-mRNA splicing, the two proteins are likely to participate in different multisubunit complexes and biological processes.