Axon-derived PACSIN1 binds to the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

Evidence that LDL receptor-related protein 1 acts as an early injury detection receptor and activates c-Jun in Schwann cells.

Schwann cells (SCs) detect injury to peripheral nerves and transform phenotypically to respond to injury and facilitate repair. Cell-signaling pathways and changes in gene expression that drive SC phenotypic transformation in injury have been described; however, the SC receptors that detect peripheral nervous system (PNS) injury have not been identified. LDL receptor-related protein 1 (LRP1) is a receptor for numerous ligands, including intracellular proteins released by injured cells and protein components of degenerated myelin. In certain cell types, including SCs, LRP1 is a cell-signaling receptor. Here, we show that binding of the LRP1 ligand, tissue-type plasminogen activator (tPA), to cultured rat SCs induces c-Jun phosphorylation, a central event in activation of the SC repair program. The response to tPA was blocked by the LRP1 antagonist, receptor-associated protein. c-Jun phosphorylation was also observed when cultured rat SCs were treated with a recombinant derivative of matrix metalloproteinase-9 that contains the LRP1 recognition motif (PEX). The ability of LRP1 to induce c-Jun phosphorylation and ERK1/2 activation was confirmed using cultures of human SCs. When tPA or PEX was injected directly into crush-injured rat sciatic nerves, c-Jun phosphorylation and ERK1/2 activation were observed in SCs in vivo. The ability of LRP1 to bind proteins released in the earliest stages of PNS injury and to induce c-Jun phosphorylation support a model in which SC LRP1 functions as an injury-detection receptor in the PNS.

Specific targeting of human caspases using designed ankyrin repeat proteins.

Abstract Caspases play important roles in cell death, differentiation, and proliferation. Due to their high homology, especially of the active site, specific targeting of a particular caspase using substrate analogues is very difficult. Although commercially available small molecules based on peptides are lacking high specificity due to overlapping cleavage motives between different caspases, they are often used as specific tools. We have selected designed ankyrin repeat proteins (DARPins) against human caspases 1-9 and identified high-affinity binders for the targeted caspases, except for caspase 4. Besides previously reported caspase-specific DARPins, we generated novel DARPins (D1.73, D5.15, D6.11, D8.1, D8.4, and D9.2) and confirmed specificity for caspases 1, 5, 6, and 8 using a subset of caspase family members. In addition, we solved the crystal structure of caspase 8 in complex with DARPin D8.4. This binder interacts with non-conserved residues on the large subunit, thereby explaining its specificity. Structural analysis of this and other previously published crystal structures of caspase/DARPin complexes depicts two general binding areas either involving active site forming loops or a surface area laterally at the large subunit of the enzyme. Both surface areas involve non-conserved surface residues of caspases.

Combined inhibition of caspase 3 and caspase 7 by two highly selective DARPins slows down cellular demise.

Caspases play important roles during apoptosis, inflammation and proliferation. The high homology among family members makes selective targeting of individual caspases difficult, which is necessary to precisely define the role of these enzymes. We have selected caspase-7-specific binders from a library of DARPins (designed ankyrin repeat proteins). The DARPins D7.18 and D7.43 bind specifically to procaspase 7 and active caspase 7, but not to other members of the family. Binding of the DARPins does not affect the active enzyme, but interferes with its activation by other caspases. The crystal structure of the caspase 7-D7.18 complex elucidates the high selectivity and the mode of inhibition. Combining these caspase-7-specific DARPins with the previously reported caspase-3-inhibitory DARPin D3.4S76R reduces the activity of caspase 3 and 7 in double-transfected HeLa cells during apoptosis. In addition, these cells showed less susceptibility to TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in living cell experiments. D7.18 and D7.43 are therefore novel tools for in vitro studies on procaspase 7 activation as well as for clarifying the role of its activation in different cellular processes. If applied in combination with D3.4S76R, they represent an excellent instrument to increase our understanding of these enzymes during various cellular processes.

Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity.

Designed ankyrin repeat proteins (DARPins) are well-established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin-target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase-7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.

Specific inhibition of caspase-3 by a competitive DARPin: molecular mimicry between native and designed inhibitors.

Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways.

HIV-1 protease inhibition potential of functionalized polyoxometalates.

Polyoxometalates (POMs) are interesting biomedical agents due to their versatile anticancer and antiviral properties, such as remarkable anti-HIV activity. Although POMs are tunable and easily accessible inorganic drug prototypes in principle, their full potential can only be tapped by enhancing their biocompatibility, for example, through organic functionalization. We have therefore investigated the HIV-1 protease inhibition potential of functionalized Keggin- and Dawson-type POMs with organic side chains. Their inhibitory performance was furthermore compared to other POM types, and the buffer dependence of the results is discussed. In addition, chemical shift mapping NMR experiments were performed to exclude POM-substrate interactions. Whereas the introduction of organic side chains into POMs is a promising approach in principle, the influence of secondary effects on the reaction system also merits detailed investigation.