npg
© 2014 Nature America, Inc. All rights reserved.
news & views active site, thus making them non-BKACE DUF849 members. The function of these non-BKACE members will be an important question in light of the enzymatic diversity present in this fold backbone. Thus, Bastard et al.1 concluded that the BKACE family represents a central scaffold with diverse members that, in total, cover a broad structural range of β-keto acids. Zhao et al.2 addressed their protein of unknown function from a structural perspective, using previously generated structures and the entire KEGG in silico docking library to perform docking prediction with an enolase-family enzyme in a genomic region of interest. This effort enriched the library for N-acylated amines, particularly N-methylated proline derivatives. An additional hint at function of the locus came from a nearby periplasmic binding protein that had a cation-π box, known for binding quaternary amines such as betaines. Docking with a refined set of betaines led to the identification of transhydroxyproline betaine (tHyp-B) as the highest-scoring ligand (Fig. 1). Modeling the active site of a nearby Rieske-type demethylase further supported tHyp-B as the substrate for this genomic region. These combined in silico structural predictions led to the hypotheses that the enolase-family enzyme HpbD was a tHyp-B racemase and that the genomic region encoded a tHyp-B catabolic pathway, which they confirmed experimentally. The HpbD racemase activity allows accumulation of tHyp-B under conditions of salt stress, where it functions as an osmoprotectant. These studies both demonstrate successful blueprints for elucidation of unknown protein function and provide fundamental insights into bacterial metabolism. In particular, Bastard et al.1
have determined that the BKACE family has a central role in the CoA-dependent metabolism of β-keto acids. The BKACE family is linked to the acetyl-CoA:CoA cycle in cells, and members either use or generate acetyl-CoA, depending on the reaction direction. Thus, when β-keto acids are being metabolized in the cell, they may affect global metabolism via alteration of post-translational lysine acetylation on metabolic and other enzymes6. The BKACE reaction fills in a void in a number of metabolic pathways, including the catabolism of the quaternary amine, carnitine. One of the links between these two stories is the functional link between carnitine, its BKACE-derived metabolite glycine betaine and hydroxyproline betaine, all of which are zwitterionic osmoprotectants. Bastard et al.1 identify a ‘G5’ family member as catalyzing the missing step in l-carnitine metabolism to glycine betaine, a potent osmoprotectant that also regulates virulence in some important opportunistic pathogens7,8. Zhao et al.2 demonstrated that the newly defined tHyp-B racemase HpbD permits preferential accumulation of metabolically inaccessible tHyp-B owing to inactivation of HpbD at high salt concentration. This demonstrates functional convergence to glycine betaine accumulation in Pseudomonas aeruginosa9 and mechanistic convergence with regulation of glycine betaine degradation in Sinorhizobium meliloti10, strengthening the case for the importance of balancing compatible solute retention and metabolism. Many bacteria accumulate osmoprotectants in response to stress and can also catabolize these same osmoprotectants. So the question can be raised: how does racemase-dependent storage differ from the
regulation of storage and use of nonchiral osmoprotectants such as glycine betaine and dimethylsulfionioproprionate? The regulatory strategies evolved by bacteria to regulate osmoprotectant homeostasis provide wonderful examples of selection driving convergent metabolic survival strategies. There is little doubt that we are just at the beginning of many scientifically lucrative projects that begin with judicious data mining, as exemplified by these two studies. These findings and other such efforts will find immediate use in informing metabolic models of bacteria and expanding the scope of building blocks for synthetic biologists. However, the real payoff will be the newly excavated biology brought to light by such methods. ■ Matthew J. Wargo is at the Department of Microbiology and Molecular Genetics, University of Vermont College of Medicine, Burlington, Vermont, USA. e-mail: [email protected] Published online 17 November 2013 doi:10.1038/nchembio.1413 References
1. Bastard, K. et al. Nat. Chem. Biol. doi:10.1038/ nchembio.1387 (17 Nov 2013). 2. Zhao, S. et al. Nature 502, 698–702 (2013). 3. Schnoes, A.M. et al. PLoS Comput. Biol. 5, e1000605 (2009). 4. Kreimeyer, A. et al. J. Biol. Chem. 282, 7191–7197 (2007). 5. de Melo-Minardi, R.C. et al. Bioinformatics 26, 3075–3082 (2010). 6. Wang, Q. et al. Science 327, 1004–1007 (2010). 7. Kapfhammer, D. et al. Appl. Environ. Microbiol. 71, 3840–3847 (2005). 8. Wargo, M.J. et al. Infect. Immun. 77, 1103–1111 (2009). 9. Fitzsimmons, L.F. et al. J. Bacteriol. 194, 4718–4726 (2012). 10. Smith, L.T. et al. J. Bacteriol. 170, 3142–3149 (1988).
Competing financial interests The author declares no competing financial interests.
WNT ACYLATION
Seeing is believing
A highly original and sensitive method using clickable bioorthogonal fatty acids and in situ proximity ligation enables the visualization of the palmitoylation of not only Wnt but also its fatty acyltransferase Porcupine in cells.
Luc G Berthiaume
U
ntil recently, progress in the study of protein palmitoylation, or the modification of proteins by the 16-carbon fatty acid palmitate, have long been impeded by the lack of proper detection methodologies. The development of the acyl biotin exchange reaction1 and
the application of the Staudinger-Bertozzi ligation and Huisgen Cu+-catalyzed alkyneazide cycloaddition to create ‘clickable’ bioorthogonal fatty acids have revolutionized how palmitoylated and fatty acylated proteins in general are detected and has enabled proteomic-type studies2. However,
nature chemical biology | VOL 10 | JANUARY 2014 | www.nature.com/naturechemicalbiology
these methods are still limited by the fact that they act on cellular proteins in a global manner. Gao and Hannoush3 now solve this problem in their report of a method capable of tracking the palmitoylated form of individual proteins in cells and its application to resolve outstanding questions 5
news & views Wnt3a C77-C88
SCoA
S2
9–12
09
O
OH
Porcupine Cell fixation Click labeling, N3
T4 ligase
O O
Phi29 polymera polymerase
O 6–9
Fluorescent nucleotides
Ser209 O
n
N N
N
Cys77-S-S-Cys88
© 2014 Nature America, Inc. All rights reserved.
npg
O n
Katie Vicari
O
N N
N
Figure 1 | Selective imaging of Wnt palmitoylation leads to a proposed model for Wnt3a processing and trafficking. In the new method (left), alkynyl-fatty acids of 13–16 carbons are added to cells, converted to their CoA derivatives and attached to proteins such as Wnt3a by protein fatty acyltransferases including Porcupine. Cells are then fixed and azido-fluorophores (such as Oregon Green488) or azido-biotin labels attached via click chemistry. Labeling with primary antibodies and then secondary antibodies conjugated to oligonucleotides provides a scaffold for a proximity ligation assay that forms a continuous circular DNA template. This template is then extended by Phi29 polymerase using red fluorescent nucleotides, creating a highly fluorescent concatemeric amplification product. By tracking the development and location of the fluorescent products (middle), Gao and Hannoush3 determined that Wnt3a, after translocation into the endoplasmic reticulum, is palmitoylated on Ser209 by Porcupine, which also promotes further glycosylation (purple) and maturation of Wnt3a. Palmitoylated Wnt3a is then targeted to MVBs and exosomes via the secretory pathway en route toward the plasma membrane for secretion.
about the identity and ordering of Wnt post-translational modifications. Wnt proteins constitute a large family of secreted molecules responsible for intercellular signaling that are involved in virtually every aspect of animal development4. Wnt proteins act locally and regionally in a concentration-dependent manner. Dysregulation of the Wnt signaling cascade has been implicated in a wide range of human diseases, including cancer4. Wnt signaling is thought to be controlled by the rate of Wnt secretion, which itself is controlled by a series of post-translational modifications. Indeed, Wnt proteins have previously been shown to be posttranslationally fatty acylated5,6 (modified by fatty acids) and glycosylated (modified by carbohydrates)7 before secretion by a highly specialized secretory machinery. Although glycosylation was demonstrated to be dispensable for Wnt activity, fatty acylation is absolutely essential. Initial MS studies on purified Wnt3a indicated that Cys77 is S-acylated by the saturated fatty acid palmitic acid, whereas Ser209 is O-acylated by the unsaturated fatty acid ω-7 palmitoleate5,6. Following translocation into the endoplasmic 6
reticulum, the prototypical Wnt3a protein was proposed to be acylated on both residues by Porcupine, a membrane-bound O-acyl transferase (MBOAT) whose precise regulation has recently been shown to be critical for physiological Wnt signaling8. However, in the recent crystal structure of Xenopus Wnt8 in complex with the mouse Frizzled-8 receptor cysteine-rich domain, only the conserved serine equivalent to Ser209 in Wnt3a is fatty acylated, whereas the conserved cysteine equivalent to Cys77 is buried in the structure and forms a disulfide bond with the Drosophila equivalent of Cys88 in Wnt3a9. Because acylation is essential for Wnt membrane binding, trafficking, secretion and receptor binding, this recent result prompted a reevaluation of the acylation status of Wnt3a at Cys77. In their paper, Gao and Hannoush3 report a new and highly sensitive method for imaging palmitoylated Wnt with subcellular resolution that uses bioorthogonal fatty acids, click chemistry and in situ proximity ligation10 in fixed cells (Fig. 1). They used the new technique to demonstrate the initial site of Wnt palmitoylation as the endoplasmic reticulum. Following palmitoylation, Wnt3a is
glycosylated and targeted to the multivesicular bodies (MVBs) via the secretory pathway en route toward exosome formation and secretion at the plasma membrane (Fig. 1). Palmitoylation of Wnt3a was independent of its glycosylation, but the extent of glycosylation of Wnt3a was promoted by the expression of either catalytically active or inactive forms of Porcupine. This suggests that Porcupine may also have a previously uncharacterized scaffolding role in the maturation of Wnt proteins. Improperly glycosylated Wnt3a was targeted for degradation. Importantly, Gao and Hannoush3 set the ‘fats’ straight on Wnt acylation by demonstrating that palmitoleic acid and other fatty acids ranging from 13–16 carbons in length can be incorporated at Ser209, but not at Cys77. Although the biological importance of the variation in fatty acid saturation and fatty acid chain length requirements for Porcupine is not clear, it may suggest that a fatty acid desaturase ‘upstream’ of Porcupine may have a role in providing the palmitoleic acid for localized Wnt targeting and secretion. Furthermore, the authors are also the first to demonstrate that the MBOAT Porcupine is S-palmitoylated on its cytosolic face and that this process is not autocatalytic but rather possibly mediated by a member of the zDHHC-PAT protein acyltransferase family. Surprisingly, the loss of Porcupine palmitoylation resulted in a slight increase in Wnt signaling, suggesting that palmitoylation might have possible negative regulatory roles that could finetune Wnt secretion. Interestingly, the new methodology developed by Gao and Hannoush3 could also be used to visualize the palmitoylation of other acylated proteins in situ and could potentially have profound effects on our understanding of the biology of these proteins inside cells. As numerous oncogenic proteins are palmitoylated, this new methodology has many possible applications that could pave the way to a better understanding of the trafficking and signaling properties of these proteins and their roles in cancer. The sensitivity and selectivity of this new method could also facilitate the screening of small molecule inhibitors of protein acyltransferases specific to known acylated proteins. These new inhibitors could eventually be useful tools to dissect the roles of palmitoylation in membrane targeting, trafficking and cell signaling. ■ Luc G. Berthiaume is at the Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada. e-mail: [email protected]
nature chemical biology | VOL 10 | JANUARY 2014 | www.nature.com/naturechemicalbiology
news & views Published online 24 November 2013; corrected online 27 November 2013 (details online); doi:10.1038/nchembio.1414 References
1. Wan, J., Roth, A.F., Bailey, A.O. & Davis, N.G. Nat. Protoc. 2, 1573–1584 (2007). 2. Hang, H.C. & Linder, M.E. Chem. Rev. 111, 6341–6358 (2011).
3. Gao, X. & Hannoush, R.N. Nat. Chem. Bio. doi:10.1038/ nchembio.1392 (24 November 2013). 4. Willert, K. & Nusse, R. Cold Spring Harb. Perspect. Biol. 4, a007864 (2012). 5. Willert, K. et al. Nature 423, 448–452 (2003). 6. Takada, R. et al. Dev. Cell 11, 791–801 (2006). 7. Komekado, H., Yamamoto, H., Chiba, T. & Kikuchi, A. Genes Cells 12, 521–534 (2007).
8. Proffitt, K.D. & Virshup, D.M. J. Biol. Chem. 287, 34167–34178 (2012). 9. Janda, C.Y., Waghray, D., Levin, A.M., Thomas, C. & Garcia, K.C. Science 337, 59–64 (2012). 10. Söderberg, O. et al. Nat. Methods 3, 995–1000 (2006).
Competing financial interests The author declares no competing financial interests.
IMMUNOLOGY
Glyco-engineering ‘super-self’
Altered glycosylation of cancer cells confers phenotypic changes that promote spread and evasion of immune responses. A new method for engineering cell surface glycans is providing insights into these mechanisms.
npg
© 2014 Nature America, Inc. All rights reserved.
Matthew S Macauley & James C Paulson
C
ancer cells exhibit changes in glycosylation that contribute to cancer progression, metastasis and evasion of immune responses1. One of the most notable changes exhibited by some cancer cells is increased expression of sialic acids on glycans of cell surface glycoproteins and glycolipids. Sialic acids are ligands for members of the sialic acid–binding immunoglobulin-like lectin (Siglec) family that are differentially expressed on white blood cells of the immune system2. Many of the Siglecs are inhibitory co-receptors that are thought to aid immune cells in ‘self ’ and ‘nonself ’ discrimination through engagement with sialic acid–containing glycans on the contacted cell2,3. This results in recruitment of the Siglec to the site of cell contact and suppression of activating receptors on the leukocyte that interact with their cognate ligands on the opposing cell. In this regard, natural killer (NK) cells are known to exhibit killing of some cancer cells that are effectively recognized as nonself, whereas other cancer cells are resistant to NK cell killing. However, the mechanisms by which some cancer cells evade killing by NK cells are not well understood. In this issue, Hudak et al.4 employ a new approach to investigate the possibility that cells expressing high levels of sialic acid are able to suppress NK cell killing by recruitment of the inhibitory receptor Siglec-7. They find that engineering cells to contain synthetic lipid-linked polymers displaying sialic acid ligands of Siglec-7 results in recruitment of the Siglec to the site of cell contact and suppression of NK cell activation. NK cells have a critical role in the immune system in destroying cells considered nonself. To aid in discrimination between self and nonself, NK cells express
a variety of inhibitory co-receptors that recognize self ligands that can suppress their activation5. Thus, if an NK cell contacts a cell with sufficient number of self ligands, these inhibitory receptors will act singly or in combination to suppress activation and cell killing. Conversely, cells that are devoid of ligands for these receptors are missing self and are susceptible to killing by NK cells if they contain ligands recognized by activating receptors. a
Among the inhibitory receptors of NK cells is Siglec-7, which can be recruited to the site of contact between NK cells and target cells containing its ligands, resulting in modulation of NK cell activation2,3,5,6. Recognizing that cancer cells with high levels of sialic acid may escape NK cell killing through suppression by Siglec-7, Hudak et al.4 set out to determine whether increasing the ligand density for this coreceptor would be sufficient to suppress
Missing self
Super self
Hyposialyated Target cell Sialic acid -7 lec Sig
Glyco-engineering
NK cell Activation
Strongly inhibited activation
Self
b
Enhanced self hypersialylated
Normal cell
Cancer cell
Increased sialylation Inhibited activation
Strongly inhibited activation
Figure 1 | Sialic acid–mediated recruitment of Siglec-7 inhibits NK cell activation. (a) Left, cells with low levels of sialic acid (hyposialylated) are considered missing self, resulting in strong activation of the NK cells. Right, glyco-engineering target cells with sialic acid polymers can transform them into super self, where the high density of ligands efficiently recruits Siglec-7 to suppress NK cell activation. (b) Left, endogenous levels of sialic acids comprise self markers on normal cells, which may engage Siglec-7 to aid in self recognition and suppress unwanted activation of NK cells. Right, many cancer cells have higher levels of sialic acid (hypersialylated), which could recruit more Siglec-7 and provide a strong inhibitory signal to the NK cells and help in immune evasion.
nature chemical biology | VOL 10 | JANUARY 2014 | www.nature.com/naturechemicalbiology
7
corrections
Nature chemical biology
erratum
Seeing is believing Luc G Berthiaume Nat. Chem. Biol. 10, 5–6 (2014); published online 24 November 2013; corrected before print 27 November 2013
npg
© 2014 Nature America, Inc. All rights reserved.
In the version of this article initially published online, the year in the publication date for reference 3 was listed as 2014 instead of 2013. The error has been corrected for the print, PDF and HTML versions of this article.
nature chemical biology
© 2014 Nature America, Inc. All rights reserved.
news & views active site, thus making them non-BKACE DUF849 members. The function of these non-BKACE members will be an important question in light of the enzymatic diversity present in this fold backbone. Thus, Bastard et al.1 concluded that the BKACE family represents a central scaffold with diverse members that, in total, cover a broad structural range of β-keto acids. Zhao et al.2 addressed their protein of unknown function from a structural perspective, using previously generated structures and the entire KEGG in silico docking library to perform docking prediction with an enolase-family enzyme in a genomic region of interest. This effort enriched the library for N-acylated amines, particularly N-methylated proline derivatives. An additional hint at function of the locus came from a nearby periplasmic binding protein that had a cation-π box, known for binding quaternary amines such as betaines. Docking with a refined set of betaines led to the identification of transhydroxyproline betaine (tHyp-B) as the highest-scoring ligand (Fig. 1). Modeling the active site of a nearby Rieske-type demethylase further supported tHyp-B as the substrate for this genomic region. These combined in silico structural predictions led to the hypotheses that the enolase-family enzyme HpbD was a tHyp-B racemase and that the genomic region encoded a tHyp-B catabolic pathway, which they confirmed experimentally. The HpbD racemase activity allows accumulation of tHyp-B under conditions of salt stress, where it functions as an osmoprotectant. These studies both demonstrate successful blueprints for elucidation of unknown protein function and provide fundamental insights into bacterial metabolism. In particular, Bastard et al.1
have determined that the BKACE family has a central role in the CoA-dependent metabolism of β-keto acids. The BKACE family is linked to the acetyl-CoA:CoA cycle in cells, and members either use or generate acetyl-CoA, depending on the reaction direction. Thus, when β-keto acids are being metabolized in the cell, they may affect global metabolism via alteration of post-translational lysine acetylation on metabolic and other enzymes6. The BKACE reaction fills in a void in a number of metabolic pathways, including the catabolism of the quaternary amine, carnitine. One of the links between these two stories is the functional link between carnitine, its BKACE-derived metabolite glycine betaine and hydroxyproline betaine, all of which are zwitterionic osmoprotectants. Bastard et al.1 identify a ‘G5’ family member as catalyzing the missing step in l-carnitine metabolism to glycine betaine, a potent osmoprotectant that also regulates virulence in some important opportunistic pathogens7,8. Zhao et al.2 demonstrated that the newly defined tHyp-B racemase HpbD permits preferential accumulation of metabolically inaccessible tHyp-B owing to inactivation of HpbD at high salt concentration. This demonstrates functional convergence to glycine betaine accumulation in Pseudomonas aeruginosa9 and mechanistic convergence with regulation of glycine betaine degradation in Sinorhizobium meliloti10, strengthening the case for the importance of balancing compatible solute retention and metabolism. Many bacteria accumulate osmoprotectants in response to stress and can also catabolize these same osmoprotectants. So the question can be raised: how does racemase-dependent storage differ from the
regulation of storage and use of nonchiral osmoprotectants such as glycine betaine and dimethylsulfionioproprionate? The regulatory strategies evolved by bacteria to regulate osmoprotectant homeostasis provide wonderful examples of selection driving convergent metabolic survival strategies. There is little doubt that we are just at the beginning of many scientifically lucrative projects that begin with judicious data mining, as exemplified by these two studies. These findings and other such efforts will find immediate use in informing metabolic models of bacteria and expanding the scope of building blocks for synthetic biologists. However, the real payoff will be the newly excavated biology brought to light by such methods. ■ Matthew J. Wargo is at the Department of Microbiology and Molecular Genetics, University of Vermont College of Medicine, Burlington, Vermont, USA. e-mail: [email protected] Published online 17 November 2013 doi:10.1038/nchembio.1413 References
1. Bastard, K. et al. Nat. Chem. Biol. doi:10.1038/ nchembio.1387 (17 Nov 2013). 2. Zhao, S. et al. Nature 502, 698–702 (2013). 3. Schnoes, A.M. et al. PLoS Comput. Biol. 5, e1000605 (2009). 4. Kreimeyer, A. et al. J. Biol. Chem. 282, 7191–7197 (2007). 5. de Melo-Minardi, R.C. et al. Bioinformatics 26, 3075–3082 (2010). 6. Wang, Q. et al. Science 327, 1004–1007 (2010). 7. Kapfhammer, D. et al. Appl. Environ. Microbiol. 71, 3840–3847 (2005). 8. Wargo, M.J. et al. Infect. Immun. 77, 1103–1111 (2009). 9. Fitzsimmons, L.F. et al. J. Bacteriol. 194, 4718–4726 (2012). 10. Smith, L.T. et al. J. Bacteriol. 170, 3142–3149 (1988).
Competing financial interests The author declares no competing financial interests.
WNT ACYLATION
Seeing is believing
A highly original and sensitive method using clickable bioorthogonal fatty acids and in situ proximity ligation enables the visualization of the palmitoylation of not only Wnt but also its fatty acyltransferase Porcupine in cells.
Luc G Berthiaume
U
ntil recently, progress in the study of protein palmitoylation, or the modification of proteins by the 16-carbon fatty acid palmitate, have long been impeded by the lack of proper detection methodologies. The development of the acyl biotin exchange reaction1 and
the application of the Staudinger-Bertozzi ligation and Huisgen Cu+-catalyzed alkyneazide cycloaddition to create ‘clickable’ bioorthogonal fatty acids have revolutionized how palmitoylated and fatty acylated proteins in general are detected and has enabled proteomic-type studies2. However,
nature chemical biology | VOL 10 | JANUARY 2014 | www.nature.com/naturechemicalbiology
these methods are still limited by the fact that they act on cellular proteins in a global manner. Gao and Hannoush3 now solve this problem in their report of a method capable of tracking the palmitoylated form of individual proteins in cells and its application to resolve outstanding questions 5
news & views Wnt3a C77-C88
SCoA
S2
9–12
09
O
OH
Porcupine Cell fixation Click labeling, N3
T4 ligase
O O
Phi29 polymera polymerase
O 6–9
Fluorescent nucleotides
Ser209 O
n
N N
N
Cys77-S-S-Cys88
© 2014 Nature America, Inc. All rights reserved.
npg
O n
Katie Vicari
O
N N
N
Figure 1 | Selective imaging of Wnt palmitoylation leads to a proposed model for Wnt3a processing and trafficking. In the new method (left), alkynyl-fatty acids of 13–16 carbons are added to cells, converted to their CoA derivatives and attached to proteins such as Wnt3a by protein fatty acyltransferases including Porcupine. Cells are then fixed and azido-fluorophores (such as Oregon Green488) or azido-biotin labels attached via click chemistry. Labeling with primary antibodies and then secondary antibodies conjugated to oligonucleotides provides a scaffold for a proximity ligation assay that forms a continuous circular DNA template. This template is then extended by Phi29 polymerase using red fluorescent nucleotides, creating a highly fluorescent concatemeric amplification product. By tracking the development and location of the fluorescent products (middle), Gao and Hannoush3 determined that Wnt3a, after translocation into the endoplasmic reticulum, is palmitoylated on Ser209 by Porcupine, which also promotes further glycosylation (purple) and maturation of Wnt3a. Palmitoylated Wnt3a is then targeted to MVBs and exosomes via the secretory pathway en route toward the plasma membrane for secretion.
about the identity and ordering of Wnt post-translational modifications. Wnt proteins constitute a large family of secreted molecules responsible for intercellular signaling that are involved in virtually every aspect of animal development4. Wnt proteins act locally and regionally in a concentration-dependent manner. Dysregulation of the Wnt signaling cascade has been implicated in a wide range of human diseases, including cancer4. Wnt signaling is thought to be controlled by the rate of Wnt secretion, which itself is controlled by a series of post-translational modifications. Indeed, Wnt proteins have previously been shown to be posttranslationally fatty acylated5,6 (modified by fatty acids) and glycosylated (modified by carbohydrates)7 before secretion by a highly specialized secretory machinery. Although glycosylation was demonstrated to be dispensable for Wnt activity, fatty acylation is absolutely essential. Initial MS studies on purified Wnt3a indicated that Cys77 is S-acylated by the saturated fatty acid palmitic acid, whereas Ser209 is O-acylated by the unsaturated fatty acid ω-7 palmitoleate5,6. Following translocation into the endoplasmic 6
reticulum, the prototypical Wnt3a protein was proposed to be acylated on both residues by Porcupine, a membrane-bound O-acyl transferase (MBOAT) whose precise regulation has recently been shown to be critical for physiological Wnt signaling8. However, in the recent crystal structure of Xenopus Wnt8 in complex with the mouse Frizzled-8 receptor cysteine-rich domain, only the conserved serine equivalent to Ser209 in Wnt3a is fatty acylated, whereas the conserved cysteine equivalent to Cys77 is buried in the structure and forms a disulfide bond with the Drosophila equivalent of Cys88 in Wnt3a9. Because acylation is essential for Wnt membrane binding, trafficking, secretion and receptor binding, this recent result prompted a reevaluation of the acylation status of Wnt3a at Cys77. In their paper, Gao and Hannoush3 report a new and highly sensitive method for imaging palmitoylated Wnt with subcellular resolution that uses bioorthogonal fatty acids, click chemistry and in situ proximity ligation10 in fixed cells (Fig. 1). They used the new technique to demonstrate the initial site of Wnt palmitoylation as the endoplasmic reticulum. Following palmitoylation, Wnt3a is
glycosylated and targeted to the multivesicular bodies (MVBs) via the secretory pathway en route toward exosome formation and secretion at the plasma membrane (Fig. 1). Palmitoylation of Wnt3a was independent of its glycosylation, but the extent of glycosylation of Wnt3a was promoted by the expression of either catalytically active or inactive forms of Porcupine. This suggests that Porcupine may also have a previously uncharacterized scaffolding role in the maturation of Wnt proteins. Improperly glycosylated Wnt3a was targeted for degradation. Importantly, Gao and Hannoush3 set the ‘fats’ straight on Wnt acylation by demonstrating that palmitoleic acid and other fatty acids ranging from 13–16 carbons in length can be incorporated at Ser209, but not at Cys77. Although the biological importance of the variation in fatty acid saturation and fatty acid chain length requirements for Porcupine is not clear, it may suggest that a fatty acid desaturase ‘upstream’ of Porcupine may have a role in providing the palmitoleic acid for localized Wnt targeting and secretion. Furthermore, the authors are also the first to demonstrate that the MBOAT Porcupine is S-palmitoylated on its cytosolic face and that this process is not autocatalytic but rather possibly mediated by a member of the zDHHC-PAT protein acyltransferase family. Surprisingly, the loss of Porcupine palmitoylation resulted in a slight increase in Wnt signaling, suggesting that palmitoylation might have possible negative regulatory roles that could finetune Wnt secretion. Interestingly, the new methodology developed by Gao and Hannoush3 could also be used to visualize the palmitoylation of other acylated proteins in situ and could potentially have profound effects on our understanding of the biology of these proteins inside cells. As numerous oncogenic proteins are palmitoylated, this new methodology has many possible applications that could pave the way to a better understanding of the trafficking and signaling properties of these proteins and their roles in cancer. The sensitivity and selectivity of this new method could also facilitate the screening of small molecule inhibitors of protein acyltransferases specific to known acylated proteins. These new inhibitors could eventually be useful tools to dissect the roles of palmitoylation in membrane targeting, trafficking and cell signaling. ■ Luc G. Berthiaume is at the Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada. e-mail: [email protected]
nature chemical biology | VOL 10 | JANUARY 2014 | www.nature.com/naturechemicalbiology
news & views Published online 24 November 2013; corrected online 27 November 2013 (details online); doi:10.1038/nchembio.1414 References
1. Wan, J., Roth, A.F., Bailey, A.O. & Davis, N.G. Nat. Protoc. 2, 1573–1584 (2007). 2. Hang, H.C. & Linder, M.E. Chem. Rev. 111, 6341–6358 (2011).
3. Gao, X. & Hannoush, R.N. Nat. Chem. Bio. doi:10.1038/ nchembio.1392 (24 November 2013). 4. Willert, K. & Nusse, R. Cold Spring Harb. Perspect. Biol. 4, a007864 (2012). 5. Willert, K. et al. Nature 423, 448–452 (2003). 6. Takada, R. et al. Dev. Cell 11, 791–801 (2006). 7. Komekado, H., Yamamoto, H., Chiba, T. & Kikuchi, A. Genes Cells 12, 521–534 (2007).
8. Proffitt, K.D. & Virshup, D.M. J. Biol. Chem. 287, 34167–34178 (2012). 9. Janda, C.Y., Waghray, D., Levin, A.M., Thomas, C. & Garcia, K.C. Science 337, 59–64 (2012). 10. Söderberg, O. et al. Nat. Methods 3, 995–1000 (2006).
Competing financial interests The author declares no competing financial interests.
IMMUNOLOGY
Glyco-engineering ‘super-self’
Altered glycosylation of cancer cells confers phenotypic changes that promote spread and evasion of immune responses. A new method for engineering cell surface glycans is providing insights into these mechanisms.
npg
© 2014 Nature America, Inc. All rights reserved.
Matthew S Macauley & James C Paulson
C
ancer cells exhibit changes in glycosylation that contribute to cancer progression, metastasis and evasion of immune responses1. One of the most notable changes exhibited by some cancer cells is increased expression of sialic acids on glycans of cell surface glycoproteins and glycolipids. Sialic acids are ligands for members of the sialic acid–binding immunoglobulin-like lectin (Siglec) family that are differentially expressed on white blood cells of the immune system2. Many of the Siglecs are inhibitory co-receptors that are thought to aid immune cells in ‘self ’ and ‘nonself ’ discrimination through engagement with sialic acid–containing glycans on the contacted cell2,3. This results in recruitment of the Siglec to the site of cell contact and suppression of activating receptors on the leukocyte that interact with their cognate ligands on the opposing cell. In this regard, natural killer (NK) cells are known to exhibit killing of some cancer cells that are effectively recognized as nonself, whereas other cancer cells are resistant to NK cell killing. However, the mechanisms by which some cancer cells evade killing by NK cells are not well understood. In this issue, Hudak et al.4 employ a new approach to investigate the possibility that cells expressing high levels of sialic acid are able to suppress NK cell killing by recruitment of the inhibitory receptor Siglec-7. They find that engineering cells to contain synthetic lipid-linked polymers displaying sialic acid ligands of Siglec-7 results in recruitment of the Siglec to the site of cell contact and suppression of NK cell activation. NK cells have a critical role in the immune system in destroying cells considered nonself. To aid in discrimination between self and nonself, NK cells express
a variety of inhibitory co-receptors that recognize self ligands that can suppress their activation5. Thus, if an NK cell contacts a cell with sufficient number of self ligands, these inhibitory receptors will act singly or in combination to suppress activation and cell killing. Conversely, cells that are devoid of ligands for these receptors are missing self and are susceptible to killing by NK cells if they contain ligands recognized by activating receptors. a
Among the inhibitory receptors of NK cells is Siglec-7, which can be recruited to the site of contact between NK cells and target cells containing its ligands, resulting in modulation of NK cell activation2,3,5,6. Recognizing that cancer cells with high levels of sialic acid may escape NK cell killing through suppression by Siglec-7, Hudak et al.4 set out to determine whether increasing the ligand density for this coreceptor would be sufficient to suppress
Missing self
Super self
Hyposialyated Target cell Sialic acid -7 lec Sig
Glyco-engineering
NK cell Activation
Strongly inhibited activation
Self
b
Enhanced self hypersialylated
Normal cell
Cancer cell
Increased sialylation Inhibited activation
Strongly inhibited activation
Figure 1 | Sialic acid–mediated recruitment of Siglec-7 inhibits NK cell activation. (a) Left, cells with low levels of sialic acid (hyposialylated) are considered missing self, resulting in strong activation of the NK cells. Right, glyco-engineering target cells with sialic acid polymers can transform them into super self, where the high density of ligands efficiently recruits Siglec-7 to suppress NK cell activation. (b) Left, endogenous levels of sialic acids comprise self markers on normal cells, which may engage Siglec-7 to aid in self recognition and suppress unwanted activation of NK cells. Right, many cancer cells have higher levels of sialic acid (hypersialylated), which could recruit more Siglec-7 and provide a strong inhibitory signal to the NK cells and help in immune evasion.
nature chemical biology | VOL 10 | JANUARY 2014 | www.nature.com/naturechemicalbiology
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corrections
Nature chemical biology
erratum
Seeing is believing Luc G Berthiaume Nat. Chem. Biol. 10, 5–6 (2014); published online 24 November 2013; corrected before print 27 November 2013
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In the version of this article initially published online, the year in the publication date for reference 3 was listed as 2014 instead of 2013. The error has been corrected for the print, PDF and HTML versions of this article.
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