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The membrane-anchored matrix metalloprotease MT1-MMP is a potent collagenolytic enzyme with a well-established role in extracellular matrix turnover and cellular invasion into collagen-rich tissues. MT1-MMP is highly expressed in various types of cancer and has been demonstrated to be directly involved in several stages of tumor progression, including primary tumor growth, angiogenesis, invasion and metastasis. Osteosarcoma is the most common type of primary bone cancer. This disease is characterized by invasive tumor growth, leading to extensive bone destruction, and metastasis to the lungs. The tumor cells in human osteosarcoma display a strong expression of MT1-MMP, but the role of MT1-MMP in osteosarcoma progression is currently unknown. In this study, we investigated the role of MT1-MMP during various stages of osteosarcoma development. We utilized an optimized orthotopic murine osteosarcoma model and human osteosarcoma cells in which the MT1-MMP gene was knocked out using CRISPR/Cas9. We observed a strong expression of MT1-MMP in wildtype cells of both primary tumors and lung metastases, but, surprisingly, MT1-MMP deficiency did not affect primary tumor growth, bone degradation or the formation and growth of lung metastases. We therefore propose that, unlike findings reported in other cancers, tumor-expressed MT1-MMP is dispensable for all stages of osteosarcoma progression.

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. Fat is an important part of our diet. The intestines absorb fats and package them into particles called lipoproteins. After reaching the bloodstream, the fat molecules (lipids) in the lipoproteins are broken down by an enzyme called lipoprotein lipase (LPL), which is located along the surface of small blood vessels. This releases nutrients that can be used by vital tissues – mainly the heart, skeletal muscle, and adipose tissues. LPL is produced by muscle and adipose tissue, but it is quickly swept up by a protein called GPIHBP1 and shuttled to its site of action inside the blood vessels. Mutations that alter the structure of LPL or GPIHBP1 can prevent the breakdown of lipids, resulting in high levels of lipids in the blood. This can lead to inflammation in the pancreas and also increases the risk of heart attacks and strokes. Many earlier studies have examined the properties of LPL, but our understanding of GPIHBP1 has been limited, mainly because it has been difficult to purify GPIHBP1 for analysis. Using genetically altered insect cells, Mysling et al. were able to purify two different forms of GPIHBP1 – a full-length version and a shorter version that lacked a small section at the end of the molecule known as the acidic domain. This revealed that the opposite end of the molecule – called the carboxyl-terminal domain – is primarily responsible for binding LPL and anchoring it inside blood vessels. Once LPL is bound to GPIHBP1, the acidic domain of GPIHBP1 helps to stabilize LPL. If GPIHBP1’s acidic domain is missing then LPL is more susceptible to losing its structure, rendering it incapable of breaking down the lipids in the blood. Mysling et al. describe a new model for how LPL and GPIHBP1 interact that explains how specific mutations in the genes that encode these proteins interfere with the delivery of LPL to small blood vessels. In the future, this could help researchers to develop new strategies to treat people with high levels of lipids in their blood.

Ookinete invasion of the mosquito midgut is an essential step for the development of the malaria parasite in the mosquito. Invasion involves recognition between a presumed mosquito midgut receptor and an ookinete ligand. Here, we show that enolase lines the ookinete surface. An antienolase antibody inhibits oocyst development of both Plasmodium berghei and Plasmodium falciparum, suggesting that enolase may act as an invasion ligand. Importantly, we demonstrate that surface enolase captures plasminogen from the mammalian blood meal via its lysine motif (DKSLVK) and that this interaction is essential for midgut invasion, because plasminogen depletion leads to a strong inhibition of oocyst formation. Although addition of recombinant WT plasminogen to depleted serum rescues oocyst formation, recombinant inactive plasminogen does not, thus emphasizing the importance of plasmin proteolytic activity for ookinete invasion. The results support the hypothesis that enolase on the surface of Plasmodium ookinetes plays a dual role in midgut invasion: by acting as a ligand that interacts with the midgut epithelium and, further, by capturing plasminogen, whose conversion to active plasmin promotes the invasion process.

Cancer-induced bone degradation is part of the pathological process associated with both primary bone cancers, such as osteosarcoma, and bone metastases originating from, e.g., breast, prostate, and colon carcinomas. Typically, this includes a cancer-dependent hijacking of processes also occurring during physiological bone remodeling, including osteoclast-mediated disruption of the inorganic bone component and collagenolysis. Extensive research has revealed the significance of osteoclast-mediated bone resorption throughout the course of disease for both primary and secondary bone cancer. Nevertheless, cancer cells representing both primary bone cancer and bone metastasis have also been implicated directly in bone degradation. We will present and discuss observations on the contribution of osteoclasts and cancer cells in cancer-associated bone degradation and reciprocal modulatory actions between these cells. The focus of this review is osteosarcoma, but we will also include relevant observations from studies of bone metastasis. Additionally, we propose a model for cancer-associated bone degradation that involves a collaboration between osteoclasts and cancer cells and in which both cell types may directly participate in the degradation process.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients’ asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

Background: The cell surface receptor (uPAR) for urokinase plasminogen activator (uPA) is a strong prognostic marker in several types of cancer. uPA cleaves the three-domain protein uPAR(I-III) into two fragments: uPAR(I), which contains domain I; and uPAR(II-III), which contains domains II and III. Established immunoassays measure a combination of uPAR forms. Our aim was to design immunoassays for specific quantification of the individual forms of uPAR. Methods: Using appropriate combinations of epitope-mapped monoclonal antibodies (Mabs) for capture and europium-labeled detection Mabs, we designed two-site sandwich time-resolved fluorescence immunoassays (TR-FIAs): TR-FIA 1 to measure uPAR(I-III) alone; TR-FIA 2 to measure both uPAR(I-III) and uPAR(II-III); and TR-FIA 3 to measure uPAR(I). To avoid detection of uPAR(I-III) in TR-FIA 3, we used a combination of the peptide uPAR antagonist AE120 and a domain I antibody, R3. AE120 blocks the binding of R3 to uPAR(I-III). In contrast, AE120 does not interact with liberated domain I and therefore does not interfere with the binding of R3 to uPAR(I). Results: The limits of quantification (CV <20%) determined by adding the proteins to uPAR-depleted plasma were <3 pmol/L in all three assays. The interassay CVs in plasma with added analytes were <11%, and recoveries were between 93% and 105%. Cross-reactivities of purified proteins in the three TR-FIAs were no more than 4%. Studies on chymotrypsin cleavage of uPAR and size-exclusion chromatography of plasma with and without added protein further supported the specificity of the assays. Conclusions: The three novel TR-FIAs accurately quantify uPAR(I-III) alone, uPAR(I-III) together with uPAR(II-III), and uPAR(I), respectively, in biological samples, including plasma, and thus are well suited for studies of the diagnostic and prognostic value of individual uPAR forms in cancer patients.

We report the crystal structure of a soluble form of human urokinase‐type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor‐stromal microenvironment in many human cancers. The structure was solved at 2.7 Å in association with a competitive peptide inhibitor of the urokinase‐type plasminogen activator (uPA)–uPAR interaction. uPAR is composed of three consecutive three‐finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA–uPAR complex. This model reveals that the receptor‐binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA‐focalized proteolysis on the cell surface and control cell adhesion and migration.

Ly6/uPAR/α-neurotoxin domain (LU-domain) is characterized by the presence of 4-5 disulfide bonds and three flexible loops that extend from a core stacked by several conversed disulfide bonds (thus also named three-fingered protein domain). This highly structurally stable protein domain is typically a protein-binder at extracellular space. Most LU proteins contain only single LU-domain as represented by Ly6 proteins in immunology and α-neurotoxins in snake venom. For Ly6 proteins, many are expressed in specific cell lineages and in differentiation stages, and are used as markers. In this study, we report the crystal structures of the two LU-domains of human C4.4A alone and its complex with a Fab fragment of a monoclonal anti-C4.4A antibody. Interestingly, both structures showed that C4.4A forms a very compact globule with two LU-domain packed face to face. This is in contrast to the flexible nature of most LU-domain-containing proteins in mammals. The Fab combining site of C4.4A involves both LU-domains, and appears to be the binding site for AGR2, a reported ligand of C4.4A. This work reports the first structure that contain two LU-domains and provides insights on how LU-domains fold into a compact protein and interacts with ligands.

Ookinete invasion of the mosquito midgut is an essential step for the development of the malaria parasite in the mosquito. Invasion involves recognition between a presumed mosquito midgut receptor and an ookinete ligand. Here, we show that enolase lines the ookinete surface. An antienolase antibody inhibits oocyst development of both Plasmodium berghei and Plasmodium falciparum, suggesting that enolase may act as an invasion ligand. Importantly, we demonstrate that surface enolase captures plasminogen from the mammalian blood meal via its lysine motif (DKSLVK) and that this interaction is essential for midgut invasion, because plasminogen depletion leads to a strong inhibition of oocyst formation. Although addition of recombinant WT plasminogen to depleted serum rescues oocyst formation, recombinant inactive plasminogen does not, thus emphasizing the importance of plasmin proteolytic activity for ookinete invasion. The results support the hypothesis that enolase on the surface of Plasmodium ookinetes plays a dual role in midgut invasion: by acting as a ligand that interacts with the midgut epithelium and, further, by capturing plasminogen, whose conversion to active plasmin promotes the invasion process. [PUBLICATION ABSTRACT]

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycolipid-anchored receptor that is involved in focalizing plasminogen activation to the cell surface due to its high-affinity binding to the urokinase-type plasminogen activator (uPA). This chapter describes recent accomplishments in the molecular understanding of the structural biology of uPAR and its interactions with the cognate ligands, uPA and vitronectin. Furthermore, the structural basis for the pharmacological inhibition of uPAR by monoclonal antibodies, recombinant fusion proteins, and synthetic peptide antagonists are discussed. These compounds may prove valuable as drug candidates in combined intervention strategies targeting tumor invasion and metastasis.