CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation.

OBJECTIVE: To investigate the efficacy of CRISPR-Cas9 mediated editing in human chondrocytes, and to develop a genome editing approach relevant to cell-based repair. METHODS: Transfection of human articular chondrocytes (both healthy and osteoarthritic) with ribonucleoprotein complexes (RNP) containing Cas9 and a crisprRNA targeting exon2 of MMP13 was performed to assess editing efficiency and effects on MMP13 protein levels and enzymatic activity. Using spheroid cultures, protein levels of a major target of MMP13, type II collagen, were assessed by western blot and immunofluorescence. RESULTS: With an editing efficiency of 63-74%, secreted MMP13 protein levels and activity were significantly reduced (percentage decrease 34.14% without and 67.97% with IL-1ß based on median values of MMP13 enzymatic activity, N = 7) comparing non-edited with edited cell populations using an exon-targeting gRNA resulting in premature stop codons through non-homologous end joining (NHEJ). Accumulation of cartilage matrix protein type II collagen was enhanced in edited cells in spheroid culture, compared to non-edited controls. CONCLUSION: CRISPR-Cas9 mediated genome editing can be used to efficiently and reproducibly establish populations of human chondrocytes with stably reduced expression of key genes of interest without the need for clonal selection. Such an editing approach has the potential to greatly enhance current cell-based therapies for cartilage repair.

Crystal structure of PTP-SL/PTPBR7 catalytic domain: implications for MAP kinase regulation.

Protein tyrosine phosphatases PTP-SL and PTPBR7 are isoforms belonging to cytosolic membrane-associated and to receptor-like PTPs (RPTPs), respectively. They represent a new family of PTPs with a major role in activation and translocation of MAP kinases. Specifically, the complex formation between PTP-SL and ERK2 involves an unusual interaction leading to the phosphorylation of PTP-SL by ERK2 at Thr253 and the inactivating dephosphorylation of ERK2 by PTP-SL. This interaction is strictly dependent upon a kinase interaction motif (KIM) (residues 224-239) situated at the N terminus of the PTP-SL catalytic domain. We report the first crystal structure of the catalytic domain for a member of this family (PTP-SL, residues 254-549, identical with residues 361-656 of PTPBR7), providing an example of an RPTP with single cytoplasmic domain, which is monomeric, having an unhindered catalytic site. In addition to the characteristic PTP-core structure, PTP-SL has an N-terminal helix, possibly orienting the KIM motif upon interaction with the target ERK2. An unusual residue in the catalytically important WPD loop promotes formation of a hydrophobically and electrostatically stabilised clamp. This could induce increased rigidity to the WPD loop and therefore reduced catalytic activity, in agreement with our kinetic measurements. A docking model based on the PTP-SL structure suggests that, in the complex with ERK2, the phosphorylation of PTP-SL should be accomplished first. The subsequent dephosphorylation of ERK2 seems to be possible only if a conformational rearrangement of the two interacting partners takes place.

SRbeta coordinates signal sequence release from SRP with ribosome binding to the translocon.

Protein targeting to the endoplasmic reticulum (ER) membrane is regulated by three GTPases, the 54 kDa subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR). Using a soluble form of SR and an XTP-binding mutant of SRbeta, we show that SRbeta is essential for protein translocation across the ER membrane. SRbeta can be cross-linked to a 21 kDa ribosomal protein in its empty and GDP-bound state, but not when GTP is bound. GTP binding to SRbeta is required to induce signal sequence release from SRP. This is achieved by the presence of the translocon, which changes the interaction between the 21 kDa ribosomal protein and SRbeta and thereby allows SRbeta to bind GTP. We conclude that SRbeta coordinates the release of the signal sequence from SRP with the presence of the translocon.

Intramolecular interactions in protein tyrosine phosphatase RPTPmu: kinetic evidence.

The receptor-like protein tyrosine phosphatase RPTPmu contains three intracellular domains: the juxtamembrane (JM) and two phosphatase domains (D1 and D2). D1 is catalytically active in vitro. The functional roles of JM and D2 are still unclear. To find out whether and how they modulate the phosphatase activity of D1, we compared the enzymatic characteristics of two constructs, containing a truncated JM and either D1 or both phosphatase domains. p-Nitrophenyl phosphate and two peptide substrates were efficiently dephosphorylated by both constructs. The specificity constant of D1 alone was up to 50% higher. D2 induces (a) decreased K(m) values for peptide substrates, (b) decreased catalytic efficiency for these substrates, (c) shifting of the optimal pH to slightly lower values, and (d) looser binding of competitive inhibitors. These data suggest that the phosphatase activity of D1 is negatively modulated and its ligand binding capacity is sensibly modified by domain D2, having possible functional significance.