The Shwachman-Diamond Syndrome (SDS) is a disorder arising from mutations in the genes encoding for the Shwachman-Bodian-Diamond Syndrome (SBDS) protein and the GTPase known as Elongation Factor Like-1 (EFL1). Together, these proteins remove the anti-association factor eIF6 from the surface of the pre-60S ribosomal subunit to promote the formation of mature ribosomes. SBDS missense mutations can either destabilize the protein fold or affect surface epitopes. The molecular alterations resulting from the latter remain largely unknown, although some evidence suggest that binding to EFL1 may be affected. We further explored the effect of these SBDS mutations on the interaction with EFL1, and showed that all tested mutations disrupted the binding to EFL1. Binding was either severely weakened or almost abolished, depending on the assessed mutation. In higher eukaryotes, SBDS is essential for development, and lack of the protein results in early lethality. The existence of patients whose only source of SBDS consists of that with surface missense mutations highlights the importance of the interaction with EFL1 for their function. Additionally, we studied the interaction mechanism of the proteins in solution and demonstrated that binding consists of two independent and cooperative events, with domains 2⁻3 of SBDS directing the initial interaction with EFL1, followed by docking of domain 1. In solution, both proteins exhibited large flexibility and consisted of an ensemble of conformations, as demonstrated by Small Angle X-ray Scattering (SAXS) experiments.

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.

Upon inflammation, monocyte-derived macrophages (MΦ) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and efficient initial response (GM-MΦ) and a good resolution (M-MΦ) of the inflammatory process. The functional activity of polarized MΦ is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MΦ that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MΦ secretome involved in the shedding of soluble endoglin. We find that the GM-MΦ secretome contains metalloprotease 12 (MMP-12), a GM-MΦ specific marker that may account for the anti-angiogenic activity of the GM-MΦ secretome. Cell surface endoglin is present in both GM-MΦ and M-MΦ, but soluble endoglin is only detected in GM-MΦ culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MΦ and endothelial cells. These data demonstrate a direct correlation between GM-MΦ polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.

Osteoarthritis (OA) is the most common articular chronic disease. However, its current treatment is limited and mostly symptomatic. Hydrogen sulfide (H2S) is an endogenous gas with recognized physiological activities. The purpose here was to evaluate the effects of the intraarticular administration of a slow-releasing H2S compound (GYY-4137) on an OA experimental model. OA was induced in Wistar rats by the transection of medial collateral ligament and the removal of the medial meniscus of the left joint. The animals were randomized into three groups: non-treated and intraarticularly injected with saline or GYY-4137. Joint destabilization induced articular thickening (≈5% increment), the loss of joint mobility and flexion (≈12-degree angle), and increased levels of pain (≈1.5 points on a scale of 0 to 3). Animals treated with GYY-4137 presented improved motor function of the joint, as well as lower pain levels (≈75% recovery). We also observed that cartilage deterioration was attenuated in the GYY-4137 group (≈30% compared with the saline group). Likewise, these animals showed a reduced presence of pro-inflammatory mediators (cyclooxygenase-2, inducible nitric oxide synthase, and metalloproteinase-13) and lower oxidative damage in the cartilage. The increment of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) levels and Nrf-2-regulated gene expression (≈30%) in the GYY-4137 group seem to be underlying its chondroprotective effects. Our results suggest the beneficial impact of the intraarticular administration of H2S on experimental OA, showing a reduced cartilage destruction and oxidative damage, and supporting the use of slow H2S-producing molecules as a complementary treatment in OA.

The four member family of "Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators", CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.