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Peer reviewed: True, Protein tyrosine phosphatase receptor-type kappa (PTPRK) is a transmembrane receptor that links extracellular homophilic interactions to intracellular catalytic activity. Previously we showed that PTPRK promotes cell-cell adhesion by selectively dephosphorylating several cell junction regulators including the protein Afadin (Fearnley et al, 2019). Here, we demonstrate that Afadin is recruited for dephosphorylation by directly binding to the PTPRK D2 pseudophosphatase domain. We mapped this interaction to a putative coiled coil (CC) domain in Afadin that is separated by more than 100 amino acids from the substrate pTyr residue. We identify the residues that define PTP specificity, explaining how Afadin is selectively dephosphorylated by PTPRK yet not by the closely related receptor tyrosine phosphatase PTPRM. Our work demonstrates that PTP substrate specificity can be determined by protein-protein interactions distal to the active site. This explains how PTPRK and other PTPs achieve substrate specificity despite a lack of specific sequence context at the substrate pTyr. Furthermore, by demonstrating that these interactions are phosphorylation-independent and mediated via binding to a non-catalytic domain, we highlight how receptor PTPs could function as intracellular scaffolds in addition to catalyzing protein dephosphorylation.

Missense mutations in the lysosomal hydrolase β-galactocerebrosidase (GALC) account for at least 40% of known cases of Krabbe disease (KD). Most of these missense mutations are predicted to disrupt the fold of the enzyme, preventing GALC in sufficient amounts from reaching its site of action in the lysosome. The predominant central nervous system (CNS) pathology and the absence of accumulated primary substrate within the lysosome mean that strategies used to treat other lysosomal storage disorders (LSDs) are insufficient in KD, highlighting the still unmet clinical requirement for successful KD therapeutics. Pharmacological chaperone therapy (PCT) is one strategy being explored to overcome defects in GALC caused by missense mutations. In recent studies, several small-molecule inhibitors have been identified as promising chaperone candidates for GALC. This Review discusses new insights gained from these studies and highlights the importance of characterizing both the chaperone interaction and the underlying mutation to define properly a responsive population and to improve the translation of existing lead molecules into successful KD therapeutics. We also highlight the importance of using multiple complementary methods to monitor PCT effectiveness. Finally, we explore the exciting potential of using combination therapy to ameliorate disease through the use of PCT with existing therapies or with more generalized therapeutics, such as proteasomal inhibition, that have been shown to have synergistic effects in other LSDs. This, alongside advances in CNS delivery of recombinant enzyme and targeted rational drug design, provides a promising outlook for the development of KD therapeutics. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full-length human GALC and used these to monitor the trafficking and processing of GALC variants in cell-based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post-translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies.

Sphingolipids are essential components of cellular membranes and defects in their synthesis or degradation cause severe human diseases. The efficient degradation of sphingolipids in the lysosome requires lipid-binding saposin proteins and hydrolytic enzymes. The glycosphingolipid galactocerebroside is the primary lipid component of the myelin sheath and is degraded by the hydrolase β-galactocerebrosidase (GALC). This enzyme requires the saposin SapA for lipid processing and defects in either of these proteins causes a severe neurodegenerative disorder, Krabbe disease. Here we present the structure of a glycosphingolipid-processing complex, revealing how SapA and GALC form a heterotetramer with an open channel connecting the enzyme active site to the SapA hydrophobic cavity. This structure defines how a soluble hydrolase can cleave the polar glycosyl headgroups of these essential lipids from their hydrophobic ceramide tails. Furthermore, the molecular details of this interaction provide an illustration for how specificity of saposin binding to hydrolases is encoded., Lysosomal degradation of sphingolipids requires lipid-binding saposin proteins and hydrolytic enzymes. Here the authors present the crystal structure of the hydrolase β-galactocerebrosidase in complex with saposin SapA and give insights into the glycosphingolipid galactocerebroside degradation mechanism.

Sphingolipids are essential components of cellular membranes and defects in their synthesis or degradation cause severe human diseases. The efficient degradation of sphingolipids in the lysosome requires lipid-binding saposin proteins and hydrolytic enzymes. The glycosphingolipid galactocerebroside is the primary lipid component of the myelin sheath and is degraded by the hydrolase β-galactocerebrosidase (GALC). This enzyme requires the saposin SapA for lipid processing and defects in either of these proteins causes a severe neurodegenerative disorder, Krabbe disease. Here we present the structure of a glycosphingolipid-processing complex, revealing how SapA and GALC form a heterotetramer with an open channel connecting the enzyme active site to the SapA hydrophobic cavity. This structure defines how a soluble hydrolase can cleave the polar glycosyl headgroups of these essential lipids from their hydrophobic ceramide tails. Furthermore, the molecular details of this interaction provide an illustration for how specificity of saposin binding to hydrolases is encoded.