Drug-tunable multidimensional synthetic gene control using inducible degron-tagged dCas9 effectors.

The nuclease-deactivated variant of CRISPR-Cas9 proteins (dCas9) fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells. In such a system the long-term presence of a stable dCas9 effector can be a draw-back precluding the ability to switch rapidly between repressed and activated target gene expression states, imposing a static environment on the synthetic regulatory circuits in the cell. To address this issue we have generated a toolkit of conditionally degradable or stabilisable orthologous dCas9 or Cpf1 effector proteins, thus opening options for multidimensional control of functional activities through combinations of orthogonal, drug-tunable artificial transcription factors.

Characterisation of a New Family of Carboxyl Esterases with an OsmC Domain.

Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/ß hydrolase fold with an extended 'lid' region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries.

The ALS8-associated mutant VAPB(P56S) is resistant to proteolysis in neurons.

VAMP/synaptobrevin associated proteins A and B (VAPA and VAPB), are type IV membrane proteins enriched on ER and Golgi membranes. Both VAPA and B interact with cytoplasmic lipid transport proteins and cytoskeletal elements to maintain the structure and composition of ER and Golgi membranes. Truncated forms of both proteins are present in some tissues but the functional significance of this is not clear. In rodents processing of VAPA occurs in most tissues, however, truncated forms of VAPB have only been reported in brain tissue. It is demonstrated here that the extent of VAPB processing in rat increases during postnatal development and that it is restricted to neurons. The C-terminal polypeptide generated by this cleavage reaction remains associated with cell membranes, but its subcellular distribution is distinct from the full-length protein. A mutant form of VAPB is associated with a familial form of neurodegenerative disease, amyotrophic lateral sclerosis type 8. The mutant protein, VAPB(P56S) , is resistant to truncation in primary neuronal cultures, although remains sensitive to some form of proteolysis when over-expressed in HEK293 cells. These data suggest that neuronal cells have a particular requirement for VAPB proteolysis and that reduced levels of processed polypeptides may contribute to the neurodegeneration associated with amyotrophic lateral sclerosis type 8.

VAPB interacts with and modulates the activity of ATF6.

A mis-sense point mutation in the human VAPB gene is associated with a familial form of motor neuron disease that has been classified as Amyotrophic Lateral Sclerosis type VIII. Affected individuals suffer from a spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) or an atypical slowly progressing form of ALS. Mammals have two homologous VAP genes, vapA and vapB. VAPA and VAPB share 76% similar or identical amino acid residues; both are COOH-terminally anchored membrane proteins enriched on the endoplasmic reticulum. Several functions have been ascribed to VAP proteins including membrane trafficking, cytoskeleton association and membrane docking interactions for cytoplasmic factors. It is shown here that VAPA and VAPB are expressed in tissues throughout the body but at different levels, and that they are present in overlapping but distinct regions of the endoplasmic reticulum. The disease-associated mutation in VAPB, VAPB(P56S), lies within a highly conserved N-terminal region of the protein that shares extensive structural homology with the major sperm protein (MSP) from nematodes. The MSP domain of VAPA and VAPB is found to interact with the ER-localized transcription factor ATF6. Over expression of VAPB or VAPB(P56S) attenuates the activity of ATF6-regulated transcription and the mutant protein VAPB(P56S) appears to be a more potent inhibitor of ATF6 activity. These data indicate that VAP proteins interact directly with components of ER homeostatic and stress signalling systems and may therefore be parts of a previously unidentified regulatory pathway. The mis-function of such regulatory systems may contribute to the pathological mechanisms of degenerative motor neuron disease.

Fumarate reductase: structural and mechanistic insights from the catalytic reduction of 2-methylfumarate.

The soluble fumarate reductase (FR) from Shewanella frigidimarina can catalyse the reduction of 2-methylfumarate with a k(cat) of 9.0 s(-1) and a K(M) of 32 microM. This produces the chiral molecule 2-methylsuccinate. Here, we present the structure of FR to a resolution of 1.5 A with 2-methylfumarate bound at the active site. The mode of binding of 2-methylfumarate allows us to predict the stereochemistry of the product as (S)-2-methylsuccinate. To test this prediction we have analysed the product stereochemistry by circular dichroism spectroscopy and confirmed the production of (S)-2-methylsuccinate.