The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits.

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A-regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes.

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin ß2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

Structural Decoding of the Netrin-1/UNC5 Interaction and its Therapeutical Implications in Cancers.

Netrin-1 has been shown to be up-regulated in a fraction of human cancers as a mechanism to allow these tumors to escape the pro-apoptotic activity of some of its main dependence receptors, the UNC5 homologs (UNC5H). Here we identify the V-2 domain of netrin-1 to be important for its interaction with the Ig1/Ig2 domains of UNC5H2. We generate a humanized anti-netrin-1 antibody that disrupts the interaction between netrin-1 and UNC5H2 and triggers death of netrin-1-expressing tumor cells in vitro. We also present evidence that combining the anti-netrin-1 antibody with epidrugs such as decitabine could be effective in treating tumors showing no or modest netrin-1 expression. These results support that this antibody is a promising drug candidate.

Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction.

Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one ß, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1N), α-5 (hLM α-5N) and ß-3 (hLM ß-3N) originating from the short arms of the human laminin αßγ heterotrimer. Corresponding studies of the hLM α-1N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function.