Publications by authors named "Kay Grobe"

48 Publications

Dispatching plasma membrane cholesterol and Sonic Hedgehog dispatch: two sides of the same coin?

Biochem Soc Trans 2021 Sep 13. Epub 2021 Sep 13.

Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany.

Vertebrate and invertebrate Hedgehog (Hh) morphogens signal over short and long distances to direct cell fate decisions during development and to maintain tissue homeostasis after birth. One of the most important questions in Hh biology is how such Hh signaling to distant target cells is achieved, because all Hh proteins are secreted as dually lipidated proteins that firmly tether to the outer plasma membrane leaflet of their producing cells. There, Hhs multimerize into light microscopically visible storage platforms that recruit factors required for their regulated release. One such recruited release factor is the soluble glycoprotein Scube2 (Signal sequence, cubulin domain, epidermal-growth-factor-like protein 2), and maximal Scube2 function requires concomitant activity of the resistance-nodulation-division (RND) transporter Dispatched (Disp) at the plasma membrane of Hh-producing cells. Although recently published cryo-electron microscopy-derived structures suggest possible direct modes of Scube2/Disp-regulated Hh release, the mechanism of Disp-mediated Hh deployment is still not fully understood. In this review, we discuss suggested direct modes of Disp-dependent Hh deployment and relate them to the structural similarities between Disp and the related RND transporters Patched (Ptc) and Niemann-Pick type C protein 1. We then discuss open questions and perspectives that derive from these structural similarities, with particular focus on new findings that suggest shared small molecule transporter functions of Disp to deplete the plasma membrane of cholesterol and to modulate Hh release in an indirect manner.
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http://dx.doi.org/10.1042/BST20210918DOI Listing
September 2021

Conserved cholesterol-related activities of Dispatched 1 drive Sonic hedgehog shedding from the cell membrane.

J Cell Sci 2022 Mar 19;135(5). Epub 2021 Aug 19.

Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Waldeyerstrasse 15, D-48149 Münster, Germany.

The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-standing questions about this pathway include how the dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells and how the resistance-nodulation-division transporter Dispatched 1 (Disp, also known as Disp1) regulates this process. Here, we show that inactivation of Disp in Shh-expressing human cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show that cholesterol export from Disp-deficient cells is reduced, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpression of transgenic Disp or the structurally related protein Patched 1 (Ptc, also known as Ptch1; a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control.
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http://dx.doi.org/10.1242/jcs.258672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403983PMC
March 2022

Surface expression of the immunotherapeutic target G in osteosarcoma depends on cell confluency.

Cancer Rep (Hoboken) 2021 Apr 2:e1394. Epub 2021 Apr 2.

Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster, Muenster, Germany.

Background: Chimeric antigen receptor (CAR) T-cell therapy of pediatric sarcomas is challenged by the paucity of targetable cell surface antigens. A candidate target in osteosarcoma (OS) is the ganglioside G , but heterogeneous expression of G limits its value.

Aim: We aimed to identify mechanisms that upregulate G target expression in OS.

Methods And Results: G surface expression in OS cells, studied by flow cytometry, was found to vary both among and within individual OS cell lines. Pharmacological approaches, including inhibition of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) and modulation of the protein kinase C, failed to increase G expression. Instead, cell confluency was found to be associated with higher G expression levels both in monolayer cultures and in tumor spheroids. The sensitivity of OS cells to targeting by G -specific CAR T cells was compared in an in vitro cytotoxicity assay. Higher cell confluencies enhanced the sensitivity of OS cells to G -antigen specific, CAR T-cell-mediated in vitro cytolysis. Mechanistic studies revealed that confluency-dependent upregulation of G expression in OS cells is mediated by increased de novo biosynthesis, through a yet unknown mechanism.

Conclusion: Expression of G in OS cell lines is highly variable and associated with increasing cell confluency in vitro. Strategies for selective upregulation of GD2 are needed to enable effective therapeutic targeting of this antigen in OS.
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http://dx.doi.org/10.1002/cnr2.1394DOI Listing
April 2021

C-Terminal Peptide Modifications Reveal Direct and Indirect Roles of Hedgehog Morphogen Cholesteroylation.

Front Cell Dev Biol 2020 12;8:615698. Epub 2021 Jan 12.

Institute of Physiological Chemistry and Pathobiochemistry and the Cells-in-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, Münster, Germany.

Hedgehog (Hh) morphogens are involved in embryonic development and stem cell biology and, if misregulated, can contribute to cancer. One important post-translational modification with profound impact on Hh biofunction is its C-terminal cholesteroylation during biosynthesis. The current hypothesis is that the cholesterol moiety is a decisive factor in Hh association with the outer plasma membrane leaflet of producing cells, cell-surface Hh multimerization, and its transport and signaling. Yet, it is not decided whether the cholesterol moiety is directly involved in all of these processes, because their functional interdependency raises the alternative possibility that the cholesterol initiates early processes directly and that these processes can then steer later stages of Hh signaling independent of the lipid. We generated variants of the C-terminal Hh peptide and observed that these cholesteroylated peptides variably impaired several post-translational processes in producing cells and Hh biofunction in eye and wing development. We also found that substantial Hh amounts separated from cholesteroylated peptide tags and and that tagged and untagged Hh variants lacking their C-cholesterol moieties remained bioactive. Our approach thus confirms that Hh cholesteroylation is essential during the early steps of Hh production and maturation but also suggests that it is dispensable for Hh signal reception at receiving cells.
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http://dx.doi.org/10.3389/fcell.2020.615698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835520PMC
January 2021

Dally-like Is Unlike Dally in Assisting Wingless Spread.

Dev Cell 2020 09;54(5):572-573

Tissue and Organ Homeostasis, Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Nicolás Cabrera 1, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Electronic address:

Lipidated morphogens can spread within tissues to regulate cell fate during development or tissue repair. How these insoluble molecules reach distant target cells remains unclear. Reporting in Nature, McGough et al. (2020) reveal the secret of how the cell-surface proteoglycan Dally-like-protein (Dlp) promotes long-range signaling of the palmitoylated morphogen Wingless.
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http://dx.doi.org/10.1016/j.devcel.2020.08.011DOI Listing
September 2020

Cerebellar Morphology and Behavioral Profiles in Mice Lacking Heparan Sulfate Gene Function.

J Dev Biol 2020 Jul 11;8(3). Epub 2020 Jul 11.

Institute of Physiological Chemistry and Pathobiochemistry, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany.

Disruption of the Heparan sulfate (HS)-biosynthetic gene N-acetylglucosamine N-Deacetylase/N-sulfotransferase 1 () during nervous system development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. However, the role of function in adult brain physiology is less explored. Therefore, we generated mice bearing a Purkinje-cell-specific deletion in gene function by using Cre/loxP technology under the control of the Purkinje cell protein 2 (Pcp2/L7) promotor, which results in HS undersulfation. We observed that mutant mice did not show overt changes in the density or organization of Purkinje cells in the adult cerebellum, and behavioral tests also demonstrated normal cerebellar function. This suggested that postnatal Purkinje cell development and homeostasis are independent of function, or that impaired HS sulfation upon deletion of Ndst1 function may be compensated for by other Purkinje cell-expressed Ndst isoforms. To test the latter possibility, we additionally deleted the second Purkinje-cell expressed Ndst family member, Ndst2. This selectively abolished reproductive capacity of compound mutant female, but not male, mice, suggesting that ovulation, gestation, or female reproductive behavior specifically depends on Ndst-dependent HS sulfation in cells types that express Cre under Pcp2/L7 promotor control.
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http://dx.doi.org/10.3390/jdb8030013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560088PMC
July 2020

Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulfate production.

Elife 2020 06 9;9. Epub 2020 Jun 9.

Aix Marseille Univ, CNRS, IBDM, Marseille, France.

Myelin destruction is followed by resident glia activation and mobilization of endogenous progenitors (OPC) which participate in myelin repair. Here we show that in response to demyelination, mature oligodendrocytes (OLG) bordering the lesion express Ndst1, a key enzyme for heparan sulfates (HS) synthesis. Ndst1+ OLG form a belt that demarcates lesioned from intact white matter. Mice with selective inactivation of Ndst1 in the OLG lineage display increased lesion size, sustained microglia and OPC reactivity. HS production around the lesion allows Sonic hedgehog (Shh) binding and favors the local enrichment of this morphogen involved in myelin regeneration. In MS patients, Ndst1 is also found overexpressed in oligodendroglia and the number of Ndst1-expressing oligodendroglia is inversely correlated with lesion size and positively correlated with remyelination potential. Our study suggests that mature OLG surrounding demyelinated lesions are not passive witnesses but contribute to protection and regeneration by producing HS.
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http://dx.doi.org/10.7554/eLife.51735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308090PMC
June 2020

Neutralising anti-drug antibodies in Fabry disease can inhibit endothelial enzyme uptake and activity.

J Inherit Metab Dis 2020 03 14;43(2):334-347. Epub 2019 Nov 14.

Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Muenster, Muenster, Germany.

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralising anti-drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence-labelled AGAL in combination with ADA-positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD-specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre-incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA-immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre-incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient-derived AGAL-deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA-complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients.
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http://dx.doi.org/10.1002/jimd.12176DOI Listing
March 2020

Soluble Heparin and Heparan Sulfate Glycosaminoglycans Interfere with Sonic Hedgehog Solubilization and Receptor Binding.

Molecules 2019 Apr 23;24(8). Epub 2019 Apr 23.

Institute of Physiological Chemistry and Pathobiochemistry and Cells-in-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, D-48149 Münster, Germany.

Sonic hedgehog (Shh) signaling plays a tumor-promoting role in many epithelial cancers. Cancer cells produce soluble a Shh that signals to distant stromal cells that express the receptor Patched (Ptc). These receiving cells respond by producing other soluble factors that promote cancer cell growth, generating a positive feedback loop. To interfere with reinforced Shh signaling, we examined the potential of defined heparin and heparan sulfate (HS) polysaccharides to block Shh solubilization and Ptc receptor binding. We confirm in vitro and in vivo that proteolytic cleavage of the N-terminal Cardin-Weintraub (CW) amino acid motif is a prerequisite for Shh solubilization and function. Consistent with the established binding of soluble heparin or HS to the Shh CW target motif, both polysaccharides impaired proteolytic Shh processing and release from source cells. We also show that HS and heparin bind to, and block, another set of basic amino acids required for unimpaired Shh binding to Ptc receptors on receiving cells. Both modes of Shh activity downregulation depend more on HS size and overall charge than on specific HS sulfation modifications. We conclude that heparin oligosaccharide interference in the physiological roles of HS in Shh release and reception may be used to expand the field of investigation to pharmaceutical intervention of tumor-promoting Shh functions.
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http://dx.doi.org/10.3390/molecules24081607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526471PMC
April 2019

Secreted metalloproteases ADAMTS9 and ADAMTS20 have a non-canonical role in ciliary vesicle growth during ciliogenesis.

Nat Commun 2019 02 27;10(1):953. Epub 2019 Feb 27.

Department of Biomedical Engineering- ND20, Cleveland Clinic Lerner Research Institute, Cleveland, OH, 44195, USA.

Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.
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http://dx.doi.org/10.1038/s41467-019-08520-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393521PMC
February 2019

Disrupting Hedgehog Cardin-Weintraub sequence and positioning changes cellular differentiation and compartmentalization .

Development 2018 09 21;145(18). Epub 2018 Sep 21.

Institute of Physiological Chemistry and Pathobiochemistry and Cells-in-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, D-48149 Münster, Germany

Metazoan Hedgehog (Hh) morphogens are essential regulators of growth and patterning at significant distances from their source, despite being produced as N-terminally palmitoylated and C-terminally cholesteroylated proteins, which firmly tethers them to the outer plasma membrane leaflet of producing cells and limits their spread. One mechanism to overcome this limitation is proteolytic processing of both lipidated terminal peptides, called shedding, but molecular target site requirements for effective Hh shedding remained undefined. In this work, by using as a model, we show that mutagenesis of the N-terminal Cardin-Weintraub (CW) motif inactivates recombinant Hh proteins to variable degrees and, if overexpressed in the same compartment, converts them into suppressors of endogenous Hh function. , additional removal of N-palmitate membrane anchors largely restored endogenous Hh function, supporting the hypothesis that proteolytic CW processing controls Hh solubilization. Importantly, we also observed that CW repositioning impairs anterior/posterior compartmental boundary maintenance in the third instar wing disc. This demonstrates that Hh shedding not only controls the differentiation of anterior cells, but also maintains the sharp physical segregation between these receiving cells and posterior Hh-producing cells.
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http://dx.doi.org/10.1242/dev.167221DOI Listing
September 2018

Taking the Occam's Razor Approach to Hedgehog Lipidation and Its Role in Development.

J Dev Biol 2018 Jan 30;6(1). Epub 2018 Jan 30.

Institute of Physiological Chemistry and Pathobiochemistry and Cells-in-Motion Cluster of Excellence, University of Münster, D-48149 Münster, Germany.

All Hedgehog (Hh) proteins signal from producing cells to distant receiving cells despite being synthesized as N-and C-terminally lipidated, membrane-tethered molecules. To explain this paradoxical situation, over the past 15 years, several hypotheses have been postulated that tie directly into this property, such as Hh transport on cellular extensions called cytonemes or on secreted vesicles called lipophorins and exosomes. The alternative situation that tight membrane association merely serves to prevent unregulated Hh solubilization has been addressed by biochemical and structural studies suggesting Hh extraction from the membrane or proteolytic Hh release. While some of these models may act in different organisms, tissues or developmental programs, others may act together to specify Hh short- and long-range signaling in the same tissues. To test and rank these possibilities, we here review major models of Hh release and transport and hypothesize that the (bio)chemical and physical properties of firmly established, homologous, and functionally essential biochemical Hh modifications are adapted to specify and determine interdependent steps of Hh release, transport and signaling, while ruling out other steps. This is also described by the term "congruence", meaning that the logical combination of biochemical Hh modifications can reveal their true functional implications. This combined approach reveals potential links between models of Hh release and transport that were previously regarded as unrelated, thereby expanding our view of how Hhs can steer development in a simple, yet extremely versatile, manner.
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http://dx.doi.org/10.3390/jdb6010003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5875562PMC
January 2018

Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the wing.

Elife 2018 03 9;7. Epub 2018 Mar 9.

Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany.

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in as a model, we show that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions.
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http://dx.doi.org/10.7554/eLife.33033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844694PMC
March 2018

Ca coordination controls sonic hedgehog structure and its Scube2-regulated release.

J Cell Sci 2017 Oct 4;130(19):3261-3271. Epub 2017 Aug 4.

Institute of Physiological Chemistry and Pathobiochemistry and Cells-in-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, D-48149 Münster, Germany

Proteolytic processing of cell-surface-bound ligands, called shedding, is a fundamental system to control cell-cell signaling. Yet, our understanding of how shedding is regulated is still incomplete. One way to increase the processing of dual-lipidated membrane-associated Sonic hedgehog (Shh) is to increase the density of substrate and sheddase. This releases and also activates Shh by the removal of lipidated inhibitory N-terminal peptides from Shh receptor binding sites. Shh release and activation is enhanced by Scube2 [signal sequence, cubulin (CUB) domain, epidermal growth factor (EGF)-like protein 2], raising the question of how this is achieved. Here, we show that Scube2 EGF domains are responsible for specific proteolysis of the inhibitory Shh N-terminus, and that CUB domains complete the process by reversing steric masking of this peptide. Steric masking, in turn, depends on Ca occupancy of Shh ectodomains, unveiling a new mode of shedding regulation at the substrate level. Importantly, Scube2 uncouples processing of Shh peptides from their lipid-mediated juxtamembrane positioning, and thereby explains the long-standing conundrum that N-terminally unlipidated Shh shows patterning activity in Scube2-expressing vertebrates, but not in invertebrates that lack Scube orthologs.
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http://dx.doi.org/10.1242/jcs.205872DOI Listing
October 2017

Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans.

J Cell Sci 2015 Jun 12;128(12):2374-85. Epub 2015 May 12.

Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, 48149 Münster, Germany Cells-in-Motion Cluster of Excellence (EXC1003-CiM), University of Münster, 48149 Münster, Germany

All Hedgehog morphogens are released from producing cells, despite being synthesized as N- and C-terminally lipidated molecules, a modification that firmly tethers them to the cell membrane. We have previously shown that proteolytic removal of both lipidated peptides, called shedding, releases bioactive Sonic hedgehog (Shh) morphogens from the surface of transfected Bosc23 cells. Using in vivo knockdown together with in vitro cell culture studies, we now show that glypican heparan sulfate proteoglycans regulate this process, through their heparan sulfate chains, in a cell autonomous manner. Heparan sulfate specifically modifies Shh processing at the cell surface, and purified glycosaminoglycans enhance the proteolytic removal of N- and C-terminal Shh peptides under cell-free conditions. The most likely explanation for these observations is direct Shh processing in the extracellular compartment, suggesting that heparan sulfate acts as a scaffold or activator for Shh ligands and the factors required for their turnover. We also show that purified heparan sulfate isolated from specific cell types and tissues mediates the release of bioactive Shh from pancreatic cancer cells, revealing a previously unknown regulatory role for these versatile molecules in a pathological context.
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http://dx.doi.org/10.1242/jcs.170670DOI Listing
June 2015

Sweet on Hedgehogs: regulatory roles of heparan sulfate proteoglycans in Hedgehog-dependent cell proliferation and differentiation.

Curr Protein Pept Sci 2015 ;16(1):66-76

Institute for Physiological Chemistry and Pathobiochemistry, Westfalische Wilhelms Universitat Munster, Waldeyerstr. 15, D-48149 Munster, Germany.

Morphogens exert their effects over long distances, typically by spreading from cell to cell to activate signal transduction in surrounding tissues in concentration-dependent manner. One example of a morphogen is the signaling molecule Hedgehog (Hh), which controls growth and patterning during development and has also been implicated in the progression of numerous cancers. To this end, accessory mechanisms that release, transport, and receive Hhs are required to elicit temporally and spatially specific responses in cells and tissues. The Hh spreading mechanism is especially intriguing, because all Hhs are released from the producing cells despite being synthesized as dually lipidated, membrane-tethered molecules. In addition to this cellular association, Hhs bind strongly to extracellular heparan sulfate proteoglycans (HSPGs), which is expected to further reduce their spreading. Paradoxically, several lines of evidence suggest that Hh gradient formation actually requires HSPG expression, and that HSPGs act as both positive and negative regulators of Hh function. This article reviews the multiple roles that HSPGs play in Hh morphogen function, and discusses their congruity with proposed mechanisms of Hh solubilization, transport, and signal reception in vertebrate and invertebrate tissues.
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http://dx.doi.org/10.2174/1389203716666150213162649DOI Listing
October 2015

Mouse macrophages completely lacking Rho subfamily GTPases (RhoA, RhoB, and RhoC) have severe lamellipodial retraction defects, but robust chemotactic navigation and altered motility.

J Biol Chem 2014 Oct 11;289(44):30772-30784. Epub 2014 Sep 11.

Institut für Molekulare Zellbiologie, Wilhelms-Universität Münster, 48149 Münster, Germany,. Electronic address:

RhoA is thought to be essential for coordination of the membrane protrusions and retractions required for immune cell motility and directed migration. Whether the subfamily of Rho (Ras homolog) GTPases (RhoA, RhoB, and RhoC) is actually required for the directed migration of primary cells is difficult to predict. Macrophages isolated from myeloid-restricted RhoA/RhoB (conditional) double knock-out (dKO) mice did not express RhoC and were essentially "pan-Rho"-deficient. Using real-time chemotaxis assays, we found that retraction of the trailing edge was dissociated from the advance of the cell body in dKO cells, which developed extremely elongated tails. Surprisingly, velocity (of the cell body) was increased, whereas chemotactic efficiency was preserved, when compared with WT macrophages. Randomly migrating RhoA/RhoB dKO macrophages exhibited multiple small protrusions and developed large "branches" due to impaired lamellipodial retraction. A mouse model of peritonitis indicated that monocyte/macrophage recruitment was, surprisingly, more rapid in RhoA/RhoB dKO mice than in WT mice. In comparison with dKO cells, the phenotypes of single RhoA- or RhoB-deficient macrophages were mild due to mutual compensation. Furthermore, genetic deletion of RhoB partially reversed the motility defect of macrophages lacking the RhoGAP (Rho GTPase-activating protein) myosin IXb (Myo9b). In conclusion, the Rho subfamily is not required for "front end" functions (motility and chemotaxis), although both RhoA and RhoB are involved in pulling up the "back end" and resorbing lamellipodial membrane protrusions. Macrophages lacking Rho proteins migrate faster in vitro, which, in the case of the peritoneum, translates to more rapid in vivo monocyte/macrophage recruitment.
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http://dx.doi.org/10.1074/jbc.M114.563270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215254PMC
October 2014

Signaling domain of Sonic Hedgehog as cannibalistic calcium-regulated zinc-peptidase.

PLoS Comput Biol 2014 Jul 17;10(7):e1003707. Epub 2014 Jul 17.

Research Group Bioinformatics, Faculty of Biology, Center of Medical Biotechnology, University of Duisburg-Essen, Essen, Germany.

Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined Zn2+ center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double-Ca2+ center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on pH.
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http://dx.doi.org/10.1371/journal.pcbi.1003707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102407PMC
July 2014

Mutational analysis of the pumpkin (Cucurbita maxima) phloem exudate lectin, PP2 reveals Ser-104 is crucial for carbohydrate binding.

Biochem Biophys Res Commun 2014 Jul 17;450(1):622-7. Epub 2014 Jun 17.

School of Chemistry, University of Hyderabad, Hyderabad 500046, India. Electronic address:

The pumpkin phloem lectin (PP2) is an RNA-binding, defense-related, chitooligosaccharide-specific, homodimeric lectin of Mr 48 kDa expressed at high concentrations in the sieve elements and companion cells of pumpkin (Cucurbita maxima). In the present study, PP2 was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway as a prerequisite for unimpaired folding and posttranslational glycosylation of recombinant PP2. Previous computational modeling and ligand docking studies predicted a putative chitooligosaccharide-binding site on the PP2 surface, which was divided into three subsites, with two amino acid residues in each subsite identified as possible candidates for interaction with chitooligosaccharides (CHOs). In this work, mutational analysis and hemagglutination assays were employed to verify the role of the predicted residues in the carbohydrate binding activity of the protein. The results obtained revealed that mutation of Ser-104 to Ala (S104A) at subsite-2 resulted in about 90% loss of agglutination activity of the protein, indicating that Ser-104 is crucial for the binding of CHOs to PP2. Also, L100A (at subsite-1) and K200A (at subsite-3) independently decreased the lectin activity by about 40%, indicating that these two residues also contribute significantly to sugar binding by PP2. Together, these findings confirm that all the three subsites contribute to varying degrees toward PP2-carbohydrate interaction, and confirm the validity of the computational model, as proposed earlier.
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http://dx.doi.org/10.1016/j.bbrc.2014.06.024DOI Listing
July 2014

Role of heparan sulfate proteoglycans in optic disc and stalk morphogenesis.

Dev Dyn 2014 Oct 6;243(10):1310-6. Epub 2014 May 6.

Department of Ophthalmology, Glick Eye Institute, Indiana University School of Medicine, Indianapolis, Indiana.

Background: Heparan sulfate proteoglycans (HSPG) are important for embryonic development by means of the regulation of gradient formation and signaling of multiple growth factors and morphogens. Previous studies have shown that Bmp/Shh/Fgf signaling are required for the regionalization of the optic vesicle (OV) and for the closure of the optic fissure (OF), the disturbance of which underlie ocular anomalies such as microphthalmia, coloboma, and optic nerve hypoplasia.

Results: To study HSPG-dependent coordination of these signaling pathways during mammalian visual system development, we have generated a series of OV-specific mutations in the heparan sulfate (HS) N-sulfotransferase genes (Ndst1 and Ndst2) and HS O-sulfotransferase genes (Hs2st, Hs6st1, and Hs6st2) in mice. Of interest, the resulting HS undersulfation still allowed for normal retinal neurogenesis and optic fissure closure, but led to defective optic disc and stalk development. The adult mutant animals further developed optic nerve aplasia/hypoplasia and displayed retinal degeneration. We observed that MAPK/ERK signaling was down-regulated in Ndst mutants, and consistent with this, HS-related optic nerve morphogenesis defects in mutant mice could partially be rescued by constitutive Kras activation.

Conclusions: These results suggest that HSPGs, depending on their HS sulfation pattern, regulate multiple signaling pathways in optic disc and stalk morphogenesis.
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http://dx.doi.org/10.1002/dvdy.24142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177358PMC
October 2014

Dendritic cell motility and T cell activation requires regulation of Rho-cofilin signaling by the Rho-GTPase activating protein myosin IXb.

J Immunol 2014 Apr 19;192(8):3559-68. Epub 2014 Mar 19.

Institute of Molecular Cell Biology, Westfalian Wilhelms University-Münster, Münster 48149, Germany.

Directed migration of stimulated dendritic cells (DCs) to secondary lymphoid organs and their interaction with Ag-specific T cells is a prerequisite for the induction of primary immune responses. In this article, we show that murine DCs that lack myosin IXB (Myo9b), a motorized negative regulator of RhoA signaling, exhibit increased Rho signaling activity and downstream acto-myosin contractility, and inactivation of the Rho target protein cofilin, an actin-depolymerizing factor. On a functional level, Myo9b(-/-) DCs showed impaired directed migratory activity both in vitro and in vivo. Moreover, despite unaltered Ag presentation and costimulatory capabilities, Myo9b(-/-) DCs were poor T cell stimulators in vitro in a three-dimensional collagen matrix and in vivo, associated with altered DC-T cell contact dynamics and T cell polarization. Accordingly, Myo9b(-/-) mice showed an attenuated ear-swelling response in a model of contact hypersensitivity. The impaired migratory and T cell stimulatory capacity of Myo9b(-/-) DCs was restored in large part by pharmacological activation of cofilin. Taken together, these results identify Myo9b as a negative key regulator of the Rho/RhoA effector Rho-kinase [Rho-associated coiled-coil-forming kinase (ROCK)]/LIM domain kinase signaling pathway in DCs, which controls cofilin inactivation and myosin II activation and, therefore may control, in part, the induction of adaptive immune responses.
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http://dx.doi.org/10.4049/jimmunol.1300695DOI Listing
April 2014

Scube2 enhances proteolytic Shh processing from the surface of Shh-producing cells.

J Cell Sci 2014 Apr 12;127(Pt 8):1726-37. Epub 2014 Feb 12.

The Institute for Physiological Chemistry and Pathobiochemistry, Westfälische Wilhelms Universität Münster, Waldeyerstrasse 15, D-48149 Münster, Germany.

All morphogens of the Hedgehog (Hh) family are synthesized as dual-lipidated proteins, which results in their firm attachment to the surface of the cell in which they were produced. Thus, Hh release into the extracellular space requires accessory protein activities. We suggested previously that the proteolytic removal of N- and C-terminal lipidated peptides (shedding) could be one such activity. More recently, the secreted glycoprotein Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like protein 2) was also implicated in the release of Shh from the cell membrane. This activity strictly depended on the CUB domains of Scube2, which derive their name from the complement serine proteases and from bone morphogenetic protein-1/tolloid metalloproteinases (C1r/C1s, Uegf and Bmp1). CUB domains function as regulators of proteolytic activity in these proteins. This suggested that sheddases and Scube2 might cooperate in Shh release. Here, we confirm that sheddases and Scube2 act cooperatively to increase the pool of soluble bioactive Shh, and that Scube2-dependent morphogen release is unequivocally linked to the proteolytic processing of lipidated Shh termini, resulting in truncated soluble Shh. Thus, Scube2 proteins act as protease enhancers in this setting, revealing newly identified Scube2 functions in Hh signaling regulation.
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http://dx.doi.org/10.1242/jcs.137695DOI Listing
April 2014

A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function.

Matrix Biol 2014 Apr 18;35:91-102. Epub 2014 Jan 18.

Insitute of Physiological Chemistry and Pathobiochemistry, Waldeyerstr. 15, University Hospital Münster, University of Münster, D-48149 Münster, Germany. Electronic address:

Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn(-/-)) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn(-/-) and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn(-/-) skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn(-/-) CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn(-/-) CS/DS. To better delineate the role of decorin, we used a 3D Dcn(-/-) fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn(-/-) fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn(-/-) fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn(-/-) samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in a concentration dependent manner unlike the Dcn(-/-) CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of Dcn(-/-) CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and keratinocytes showed that, unlike Dcn(-/-) CS/DS, wild-type CS/DS promoted differentiation of keratinocytes. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn(-/-) mice and possibly Ehlers-Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence keratinocyte behavior.
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http://dx.doi.org/10.1016/j.matbio.2014.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165699PMC
April 2014

Heparan sulfate expression in the neural crest is essential for mouse cardiogenesis.

Matrix Biol 2014 Apr 5;35:253-65. Epub 2013 Nov 5.

Institut für Physiologische Chemie und Pathobiochemie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany; Institut für Molekulare Zellbiologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany. Electronic address:

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1(-/-) embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect.
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http://dx.doi.org/10.1016/j.matbio.2013.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324438PMC
April 2014

Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. [corrected].

Cell Stem Cell 2013 Apr;12(4):426-39

Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, D-80336, Germany.

As a result of brain injury, astrocytes become activated and start to proliferate in the vicinity of the injury site. Recently, we had demonstrated that these reactive astrocytes, or glia, can form self-renewing and multipotent neurospheres in vitro. In the present study, we demonstrate that it is only invasive injury, such as stab wounding or cerebral ischemia, and not noninvasive injury conditions, such as chronic amyloidosis or induced neuronal death, that can elicit this increase in plasticity. Furthermore, we find that Sonic hedgehog (SHH) is the signal that acts directly on the astrocytes and is necessary and sufficient to elicit the stem cell response both in vitro and in vivo. These findings provide a molecular basis for how cells with neural stem cell lineage emerge at sites of brain injury and imply that the high levels of SHH known to enter the brain from extraneural sources after invasive injury can trigger this response.
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http://dx.doi.org/10.1016/j.stem.2013.01.019DOI Listing
April 2013

An emerging role of Sonic hedgehog shedding as a modulator of heparan sulfate interactions.

J Biol Chem 2012 Dec 1;287(52):43708-19. Epub 2012 Nov 1.

Institute for Physiological Chemistry and Pathobiochemistry, University Hospital Münster, Waldeyerstrasse 15, D-48149 Münster, Germany.

Major developmental morphogens of the Hedgehog (Hh) family act at short range and long range to direct cell fate decisions in vertebrate and invertebrate tissues. To this end, Hhs are released from local sources and act at a distance on target cells that express the Hh receptor Patched. However, morphogen secretion and spreading are not passive processes because all Hhs are synthesized as dually (N- and C-terminal) lipidated proteins that firmly tether to the surface of producing cells. On the cell surface, Hhs associate with each other and with heparan sulfate (HS) proteoglycans. This raises the question of how Hh solubilization and spreading is achieved. We recently discovered that Sonic hedgehog (Shh) is solubilized by proteolytic processing (shedding) of lipidated peptide termini in vitro. Because unprocessed N termini block Patched receptor binding sites in the cluster, we further suggested that their proteolytic removal is required for simultaneous Shh activation. In this work we confirm inactivity of unprocessed protein clusters and demonstrate restored biological Shh function upon distortion or removal of N-terminal amino acids and peptides. We further show that N-terminal Shh processing targets and inactivates the HS binding Cardin-Weintraub (CW) motif, resulting in soluble Shh clusters with their HS binding capacities strongly reduced. This may explain the ability of Shh to diffuse through the HS-containing extracellular matrix, whereas other HS-binding proteins are quickly immobilized. Our in vitro findings are supported by the presence of CW-processed Shh in murine brain samples, providing the first in vivo evidence for Shh shedding and subsequent solubilization of N-terminal-truncated proteins.
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http://dx.doi.org/10.1074/jbc.M112.356667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527956PMC
December 2012

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development.

Development 2012 Aug 28;139(15):2730-9. Epub 2012 Jun 28.

Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

Glycosaminoglycans (GAGs) play a central role in embryonic development by regulating the movement and signaling of morphogens. We have previously demonstrated that GAGs are the co-receptors for Fgf10 signaling in the lacrimal gland epithelium, but their function in the Fgf10-producing periocular mesenchyme is still poorly understood. In this study, we have generated a mesenchymal ablation of UDP-glucose dehydrogenase (Ugdh), an essential biosynthetic enzyme for GAGs. Although Fgf10 RNA is expressed normally in the periocular mesenchyme, Ugdh mutation leads to excessive dispersion of Fgf10 protein, which fails to elicit an FGF signaling response or budding morphogenesis in the presumptive lacrimal gland epithelium. This is supported by genetic rescue experiments in which the Ugdh lacrimal gland defect is ameliorated by constitutive Ras activation in the epithelium but not in the mesenchyme. We further show that lacrimal gland development requires the mesenchymal expression of the heparan sulfate N-sulfation genes Ndst1 and Ndst2 but not the 6-O and 2-O-sulfation genes Hs6st1, Hs6st2 and Hs2st. Taken together, these results demonstrate that mesenchymal GAG controls lacrimal gland induction by restricting the diffusion of Fgf10.
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http://dx.doi.org/10.1242/dev.079236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392702PMC
August 2012

The amino acid tryptophan prevents the biosynthesis of dermatan sulfate.

Mol Biosyst 2011 Oct 26;7(10):2872-81. Epub 2011 Jul 26.

Institute of Physiological Chemistry and Pathobiochemistry, University Hospital Münster, 48149 Münster, Germany.

An important part of the biosynthesis of proteoglycans is the epimerization of glycosaminoglycan chains. As a consequence of the conversion of chondroitin sulfate (CS) to dermatan sulfate (DS), the glycosaminoglycans become more flexible and enable DS to perform more sophisticated signaling functions. In a recent study, we generated a chimera (S222A) composed of a truncated form of a DS (decorin) and CS (CSF-1) containing proteoglycan and analyzed the influence of the core protein on the extent of epimerization. C-terminal truncation constructs from S222A enabled us to identify an amino acid segment that lies within the CSF-1 part which prevents DS synthesis. Co-localization experiments using S222A-HA and DCN-Flag showed different intracellular localizations for the proteoglycans during biosynthesis. A data base search revealed a sequence motif (TNWVP) within the CSF-1 moiety that is found to be important in other proteoglycans. A single substitution of tryptophan-216 to leucine (W216L) in the chimera S222A increased the amount of l-IdoA to 12-16%. Co-localization with an ER-marker demonstrated that the biosynthesis of recombinant decorin is similar to the chimera S222A and S222A(W216L) in HEK293 cells. Co-staining of S222A-HA and S222A(W216L)-Flag showed different intracellular localizations for the proteoglycans. A more detailed analysis of the glycosaminoglycans reflects a similar total sulfate content for S222A and S222A(W216L). The 4/6 sulfation ratio was similar for decorin and S222A, but altered for S222A(W216L). However, the binding of fibroblasts growth factor-1 to CS/DS was only partially dependent on epimerization. These results are consistent with the model in which the core protein, via the amino acid tryptophan, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase.
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http://dx.doi.org/10.1039/c1mb05139cDOI Listing
October 2011

Sonic hedgehog shedding results in functional activation of the solubilized protein.

Dev Cell 2011 Jun;20(6):764-74

Institute for Physiological Chemistry and Pathobiochemistry, University Hospital Münster, Germany.

All Hedgehog (Hh) proteins are released from producing cells despite being synthesized as N- and C-terminally lipidated, membrane-tethered molecules. Thus, a cellular mechanism is needed for Hh solubilization. We previously suggested that a disintegrin and metalloprotease (ADAM)-mediated shedding of Sonic hedgehog (ShhNp) from its lipidated N and C termini results in protein solubilization. This finding, however, seemed at odds with the established role of N-terminal palmitoylation for ShhNp signaling activity. We now resolve this paradox by showing that N-palmitoylation of ShhNp N-terminal peptides is required for their proteolytic removal during solubilization. These peptides otherwise block ShhNp zinc coordination sites required for ShhNp binding to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp function. We suggest a functional model in which membrane-tethered multimeric ShhNp is at least partially autoinhibited in trans but is processed into fully active, soluble multimers upon palmitoylation-dependent cleavage of inhibitory N-terminal peptides.
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http://dx.doi.org/10.1016/j.devcel.2011.05.010DOI Listing
June 2011
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