Periodic Reporting for period 2 - ChondUb (Identification and characterisation of novel WWP2 substrates and their role in chondrogenesis and osteoarthritis)
Reporting period: 2017-07-01 to 2018-06-30
The age of the population in the EU and worldwide increases continuously, therefore understanding of the molecular processes involved in the pathogenesis and progression of OA is of great importance.
A network of proteins controlled by regulatory proteins is responsible for the proper functioning of living organisms. Ubiquitination is a process where a small protein is attached to other proteins and causes their breakdown or changes their way of acting. Some proteins are known to regulate cartilage production and degradation. WWP2 is a protein which ubiquitinates other proteins, and is known to regulate production of cartilage (chondrogenesis).
We aimed to identify and characterise WWP2 interactors and substrates using a combination of proteomics and molecular biology techniques. In addition, we will investigate the role of WWP2 and its substrates in cartilage biology with the goal to uncover molecular targets for OA treatment and other WWP2-related diseases.
A significant amount of time was allocated to generating tools necessary for the project to progress. Using these tools, we identified several potential WWP2 interactors/substrates. The proteins were identified by co-immunoprecipitations (Co-IPs) using the catalytically inactive WWP2 in combination with mass spectrometry. Further, several possible WWP2 substrates were identified in cells depleted of WWP2 (using siRNA) and in cells over-expressing tetracycline-inducible, active WWP2. The proteins were identified using quantitative mass spectrometry.
Five of the identified proteins were confirmed as WWP2 interactors in co-IPs and/or in-vitro binding assays. In-vitro ubiquitination assays indicate that four of the proteins are ubiquitinated by WWP2.
These five WWP2 interactors were investigated for their impact on the chondrogenesis in human mesenchymal stem cells (hMSCs). WWP2 and the interactors were depleted in hMSCs using siRNA prior to the induction of the chondrogenesis. The chondrogenesis process, resulting in cartilage pellets formation, takes 14 days to complete in our experimental conditions and most of the cartilage-specific genes peak in expression at day 7. Using real-time RT-PCR we determined the expression of the cartilage-specific genes (e.g. collagen2, aggrecan, collagen10, and Sox9). Deletion of WWP2 resulted in impairment of the chondrogenesis process. Moreover, deletion of one of the investigated interactors also had a negative impact, and deletion of another had a positive impact on the cartilage-specific gene expression and cartilage pellet formation.
In addition, we generated a novel transgenic mouse, Wwp2-C-KO, which lacks the first exon of the cartilage specific isoform of Wwp2 using CRISPR/Cas9. We performed RNA-sequencing in costal chondrocytes isolated from ribs of 7 days old mice. Deletion of the cartilage-specific isoform resulted in subtle changes at the level of gene expression.
We also evaluated the skeletal development in Wwp2-C-KO mice. We performed bone measurements, quantitative computed tomography (-QCT) scans of the skulls and histological staining on the legs; H&E to visualise tissue structure and cellular organization, and safranin ‘O’ to detect cartilage proteoglycans. Wwp2-C-KO mice showed differences in skull shape morphology along with delayed endochondral ossification.
Aspects of these results have been presented at two international meetings the 9th Cold Spring Harbor meeting on The Ubiquitin Family, Cold Spring Harbor, New York, USA; and at FASEB Science Research Conferences on Ubiquitin and Cellular Regulation, Snowmass, Colorado, USA.
Currently, we are drafting a manuscript for a scientific publication from the obtained data. In addition, we secured funding for 12 months, which will allow as to publish and apply for more funding in the future. In conclusion, our main goals were accomplished.
The contribution of the project to the challenges of Horizon 2020, in particularly the “Health, Demographic Change and Well-Being” and “Understanding disease” challenges remains of high importance.