Notum Is Required for Neural and Head Induction via Wnt Deacylation, Oxidation, and Inactivation

Zhang, Xinjun; Cheong, Seong-Moon; Amado, Nathália G.; Reis, Alice H.; MacDonald, Bryan T.; Zebisch, M.; Jones, E Y; Abreu, José G.; He, Xi

doi:10.1016/j.devcel.2015.02.014

Cited by 155 publications

(167 citation statements)

References 61 publications

(140 reference statements)

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“…S1). Hence, like Wnt proteins, for which palmitoleylation was recently shown to be reversible (34,35), fatty acid cleavage by a carboxylesterase may regulate targeting of hydrophilic porins. Indeed, such potential carboxylesterases/hydrolases with a consensus mycoloylation site have been found in C. glutamicum (Table S3).…”

Section: Discussionmentioning

confidence: 99%

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Carel

Marcoux

Réat

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.Corynebacteriales | O-acylation | sequence motif | top-down proteomics | NMR

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Section: Discussionmentioning

confidence: 99%

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Carel

Marcoux

Réat

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…S9 A and B), we compared the shape of this cavity with that present in other proteins that bind to cis-unsaturated fatty acids and regulate Wnt signaling. Notum, a Wnt deacylase, inhibits Wnt signaling by cleaving the Wnt fatty acyl moiety (36,37). Examination of the Notum structure bound to peptide-conjugated C16:1 revealed an alternative geometry of the hydrophobic cavity with a narrow base where the cis-Δ9 desaturation site was positioned, allowing the hydrocarbon chain to fold onto itself (SI Appendix, Fig.…”

Section: Wnt Regulators Recognize Cis-unsaturated Fatty Acids With Cumentioning

confidence: 99%

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Nile¹,

Mukund²,

Stanger³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

105

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Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt-FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt-FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function.nt signaling is evolutionarily conserved from metazoans to humans (1). It is critical for tissue homeostasis and during development (2, 3). On the other hand, derangements in Wnt signaling are associated with severe pathologies in mammals, including cancer (4-6). Signaling is initiated at the cell surface where the secreted, lipid-modified Wnt glycoprotein interacts with the extracellular N-terminal cysteine-rich domain (CRD) of the frizzled (FZD) receptor (7). This high-affinity interaction occurs at two distinct contact sites, one comprising a protein-fatty acyl interface and another a protein-protein interaction interface (8-10). Sequence analysis revealed that there are 10 human frizzled genes grouped into four clusters (SI Appendix, Fig. S1A).Among their multiple roles in physiology, FZD receptors have emerged as critical regulators of Wnt-dependent stem cell processes. For example, FZD7 is a critical component of the stem cell niche and was shown by genetic knockdown experiments to be essential for maintaining human embryonic and epithelial limbal stem cells in an undifferentiated state (11,12). Of the 10 mammalian frizzleds, FZDs 1, 2, and 7 are enriched at the base of mammalian adult intestinal crypts where the stem cells reside (13,14), and FZD7 is required for stem cell-mediated regeneration of the intestinal epithelium (15). Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors (16)(17)(18)(19)...

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“…Notum proteins are secreted lipases that deacylate Wnt ligands and prevent binding to Frizzled receptors (Kakugawa et al, 2015;Zhang et al, 2015), thus inhibiting Wnt signaling in many animals, including planarians, fruit flies, zebrafish and mammals (Flowers et al, 2012;Gerlitz and Basler, 2002;Giráldez et al, 2002;Petersen and Reddien, 2011;Traister et al, 2008). Planarians have a single notum homolog, and in uninjured animals, notum is expressed in the head at the anterior body pole and the anterior brain commissure ( Fig.…”

Section: Planarians Robustly Restore Brain:body Proportionality Throumentioning

confidence: 99%

Wnt/Notum spatial feedback inhibition controls neoblast differentiation to regulate reversible growth of the planarian brain

2015

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Mechanisms determining final organ size are poorly understood. Animals undergoing regeneration or ongoing adult growth are likely to require sustained and robust mechanisms to achieve and maintain appropriate sizes. Planarians, well known for their ability to undergo whole-body regeneration using pluripotent adult stem cells of the neoblast population, can reversibly scale body size over an order of magnitude by controlling cell number. Using quantitative analysis, we showed that after injury planarians perfectly restored brain:body proportion by increasing brain cell number through epimorphosis or decreasing brain cell number through tissue remodeling (morphallaxis), as appropriate. We identified a pathway controlling a brain size set-point that involves feedback inhibition between wnt11-6/wntA/wnt4a and notum, encoding conserved antagonistic signaling factors expressed at opposite brain poles. wnt11-6/wntA/wnt4a undergoes feedback inhibition through canonical Wnt signaling but is likely to regulate brain size in a non-canonical pathway independently of beta-catenin-1 and APC. Wnt/Notum signaling tunes numbers of differentiated brain cells in regenerative growth and tissue remodeling by influencing the abundance of brain progenitors descended from pluripotent stem cells, as opposed to regulating cell death. These results suggest that the attainment of final organ size might be accomplished by achieving a balance of positional signaling inputs that regulate the rates of tissue production.

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Notum Is Required for Neural and Head Induction via Wnt Deacylation, Oxidation, and Inactivation

Cited by 155 publications

References 61 publications

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Wnt/Notum spatial feedback inhibition controls neoblast differentiation to regulate reversible growth of the planarian brain

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