Biology subjects

Roelink, H.

Publications and source records attributed to Roelink, H..

5 recordsLinked to original sources

bioRxiv2020

Non-cell autonomous inhibition of the Shh pathway due to impaired cholesterol biosynthesis requires Ptch1/2

Congenital birth defects due to mutations of enzymes involved cholesterol synthesis, like Smith-Lemli-Opitz syndrome (SLOS) and Lathosterolosis are characterized by an accumulation of cholesterol precursors. The phenotype of both SLOS and Lathosterolosis have similarities to syndromes associated with abnormal Sonic hedgehog (Shh) signaling, consistent with the observation that cholesterol precursors and their derivative can inhibit Shh signaling. Two types of multipass membrane proteins play central roles in Shh signal transduction, the putative Resistance, Nodulation and Division (RND) antiporters Patched (Ptch)1 and -2, and the G-protein coupled receptor Smoothened (Smo). Sterols have been suggested as cargo for Ptch1/2, while Smo activity can affected both positively and negatively by steroidal molecules. We demonstrate that embryonic stem cells with mutations in the 7-dehydroxycholesterol reductase (7Dhcr) or sterol-C5-desaturase (Sc5d) gene reduce the Hh response in adjacent wildtype cells when grown in mosaic organoids. This non-cell autonomous inhibitory activity of the mutant cells requires the presence of both Ptch1 and Ptch2. These observations support a model in which late cholesterol precursors that accumulate in cells lacking 7Dhcr are the cargo for Ptch1 and Ptch2 efflux activity and mediate the non-cell autonomous inhibition of Smo.

developmental biology↗

bioRxiv2019

Association of Sonic Hedgehog with the Extracellular Matrix Requires its Putative Zinc-Peptidase Activity

Sonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination domain, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and thus potentially involved in the extracellular distribution of Shh. We found that the zinc-coordination fold of Shh is required for its association with ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 M zinc and is prevented by mutations affecting critical conserved catalytical residues as well as extracellular calcium. Our results demonstrate that the zinc-coordination fold is required for ECM-association and suggest that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling.

developmental biology↗

bioRxiv2018

Loss of the Heparan Sulfate Proteoglycan Glypican5 facilitates long range Shh signaling.

As a morphogen, Sonic Hedgehog (Shh) mediates signaling at a distance from its sites of synthesis. After secretion, Shh must traverse a distance through the extracellular matrix (ECM) to reach the target cells and activate the Hh response. Extracellular matrix proteins, in particular the Heparan Sulfate Proteoglycans (HSPGs) of the Glypican family have both negative and positive effects on non-cell autonomous Shh signaling, all attributed to their ability to bind Shh. Using mouse embryonic stem cell-derived mosaic tissues with compartments that lack the glycosyltransferases Exostosin1 (Ext1) and Exostosin2 (Ext2), or the HSPG core protein Glypican5 we show that cells surrounded by a mutated extracellular matrix are highly proficient distributing Shh. In contrast, cells that lack Ext1 function poorly secrete Shh. Our results confirm earlier observations that HSPGs can have both positive (Shh export) and negative influences (Shh distribution), and are supporting a model in which Shh presented on the cell surface in the context of HSPGs preferentially distributes into ECM that lacks HSPGs, possibly due to the absence of Shh sequestering molecules.

cell biology↗

bioRxiv2018

The Hedgehog signaling domain was acquired from a prokaryote.

Sonic Hedgehog (Shh) coordinates Zn2+ in a manner that resembles that of peptidases. The ability of Shh to undergo autoproteolytic processing is impaired in mutants that affect the Zn2+ coordination, while mutating residues essential for catalytic activity results in more stable forms of Shh. The residues involved in Zn2+ coordination in Shh are found mutated in some individuals with the congenital birth defect holoprosencephaly, demonstrating their importance in development. Highly conserved Shh domains are found a parts of some bacterial proteins that are members of the larger family of DD-peptidases, supporting the notion that Shh acts as a peptidase. Whereas this Hh/DD-peptidase motif is present in Hedgehog (Hh) proteins of nearly all animals, it is not present in Drosophila Hh, indicating that Hh signaling in fruit flies is derived, and perhaps not a good model for vertebrate Shh signaling. Sequence analysis of Hh proteins and their possible evolutionary precursors suggest that the evolution of modern Hh might have involved horizontal transfer of a bacterial gene coding of a Hh/DD-peptidase into a Cnidarian ancestor, recombining to give rise to modern Hh.

evolutionary biology↗

bioRxiv2017

ShhN-mediated activation of Smo in the absence of Ptch1/2 function

Sonic Hedgehog (Shh) signaling is characterized by strict non-cell autonomy; cells expressing Shh do not respond to their ligand. Here, we identify several Shh mutations that gain the ability to activate the Hedgehog (Hh) pathway in cis. This activation requires the extracellular cysteine rich domain of Smoothened, but is otherwise independent of Ptch1/2. Many of the identified mutations disrupt either a highly conserved catalytic motif found in peptidases or an a-helix domain frequently mutated in holoprosencephaly-causing SHH alleles. The expression of gain-of-function mutants often results in the accumulation of unprocessed Shh pro-peptide, a form of Shh we demonstrate is sufficient to activate the Hh response cell-autonomously. Our results demonstrate that Shh is capable of activating the Hh pathway via Smo independently of Ptch1/2, and that it harbors an intrinsic mechanism that prevents cell-autonomous activation of the pathway to favor non-cell autonomous signaling.

developmental biology↗