Dr Natalia Riobo-Del Galdo

Introduction

My lab is interested in understanding the molecular details of the complex Hedgehog (Hh) signalling network in vertebrate cells and tissues. Hedgehog proteins play a fundamental role in embryonic development and postnatal tissue homeostasis, leading to cancer initiation and progression when its activity is increased by mutations, ligand overexpression or crosstalk with other oncogenic pathways. We study the mechanisms of Hedgehog signalling using normal and cancer cells in culture or co-culture to study protein-protein interactions within cells, posttranslational modifications like phosphorylation and ubiquitylation by different biochemical methods, activation of second messengers, transcriptional changes by RNA-seq and qPCR, and luciferase reporter assays after expressing proteins with specific mutations or deletions or after editing genomes using CRISPR/Cas9 engineering. We investigate the functional consequences in a variety of cell-based assays, patient-derived samples and animals through collaboration with other groups.

Detailed research programme

Mechanistic basis of canonical and non-canonical Hedgehog signalling

Within this broad area, we have projects in the following specific aspects:

Role of PTCH1-PTCH2 interaction in shaping the responsiveness to Hh ligands. Using mutagenesis, fluorescence resonance energy transfer recovery after photobleaching (FRAP), protein-protein interaction analysis and activity assays, we are establishing the differential activities of homo and heterodimer receptors and the key domains that regulate interaction and activity.

• Modulation of PTCH1 activity by β-arrestins. Using mass spectrometry, mutagenesis, protein-protein interaction, stability and signalling to the Gli transcription factors.
  Structural and functional characterisation of phosphorylation of the intracellular domain of PTCH1 in regions associated with cancer
• Interplay of PTCH1 activity with Niemann-Pick disease proteins 1 and 2 in the regulation of autophagy and canonical Hh signalling using CRISPR/Cas9 genome editing, autophagic flux studies and confocal microscopy.
• Characterisation of the mechanism of action of the inhibitory protein Itm2a on Gli transcriptional activity.
• We are studying the mechanobiology aspects of Hh signalling during osteogenic differentiation using 3D culture on microparticles of different materials and textures in collaboration with Dr. Amer (Leeds).

Hedgehog signalling in cancer

We generated an isogenic cellular model of colon cancer with specific mutations in the Hh pathway that result in specific dysregulation of its non-canonical signalling using CRISPR/Cas9. Using this model, we are investigating the consequences of the mutations on oncogenic properties in vitro and in mice xenograft models, transcriptional changes of therapeutic targets and altered growth factor signalling pathways in collaboration with Prof. Canettieri (Italy).

Hedgehog signalling in fibrosis

Using scleroderma as a disease model of fibrosis, we are investigating the role of Hh signalling and its cell-type specific characteristics in human skin derived stromal cells from patients and its involvement in myofibroblast differentiation and activation in collaboration with Prof. Del Galdo (Leeds).

Structure-function of PTCH-domain containing proteins

In a continuing collaboration with Prof. Goldman (Finland), we are investigating the structure of PTCHD1, a protein associated with autism spectrum disorders, by cryo-EM and its role as cholesterol transporter using a series of biochemical assays.