Biology subjects

Rossolillo, P.

Publications and source records attributed to Rossolillo, P..

3 recordsLinked to original sources

bioRxiv2023

Abca4 inhibition in a cone-rich rodent leads to Stargardt Disease type 1-like retinal degeneration

Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for the most common form of inherited macular degeneration known as Stargardt Disease type 1 (STGD1). STGD1 typically declares early in life and leads to severe visual handicap. Abca4 gene deletion mouse models of STGD1 show increased accumulation of lipofuscin, a hallmark of the disease, but unlike the human disease show mostly no photoreceptor degeneration or functional decline (an albino Abca4-/- mouse exhibits photoreceptor degeneration although functional parameters were not studied). Reasoning that the small cone population of mice (<3%) might compromise more faithful modelling of human maculopathies, we performed subretinal injections of CRISPR/Cas9-Abca4 recombinant Adeno-Associated Virus constructs into young Fat Sand Rats (Psammomys obesus), a diurnal rodent containing >30% cones. Sanger sequencing of the CRISPR-targeted sequence showed clear edition of the Abca4 gene. At 2 months post- injection, non-invasive fundus imaging showed widespread photoreceptor loss, confirmed by optical coherence tomography. Functional recording by scotopic and photopic single flash, and photopic flicker electroretinography, showed significant decline in photopic (cone) but not scotopic (rod) light responses. Post-mortem real-time PCR, immunohistochemistry and western blotting showed significant decrease of cone-specific (MW cone opsin) but not rod- specific (rhodopsin) markers. Transmission electron microscopy showed large numbers of lipid inclusions in treated but not control retinal pigmented epithelium. Finally, ultrahigh performance liquid chromatographic analysis of whole P. obesus eyes showed the presence of all-trans retinal-dimer, also seen in Abca4-/- mice but not normal rod-rich mouse or rat eyes. In conclusion, this animal model of STGD1 more accurately reflects human STGD1 and should be valuable for characterizing pathogenic pathways and exploring treatment options.

pathology↗

bioRxiv2022

In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2

Cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is a prominent example within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis, interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2. We also describe pH-dependent conformational changes of BicD2 using cryoEM, template-free structure predictions, and biophysical tools. Our results will help define the biochemical parameters for the in vitro reconstitution of higher order BicD2 protein complexes.

biochemistry↗

bioRxiv2020

Genetically flexible but conserved: a new essential motif in the C-ter domain of HIV-1 group M integrases

Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. IMPORTANCEIntensive studies on HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatments escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens on the possibility of adapting to the optimisation of further functionalities exerted by the same protein. Such property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

molecular biology↗