ABSTRACT
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.
Subject(s)
Cysteine/chemistry , Fatty Acids, Unsaturated/chemistry , Frizzled Receptors/chemistry , Wnt Proteins/chemistry , beta Catenin/chemistry , Crystallography, X-Ray , Cysteine/metabolism , Fatty Acids, Unsaturated/metabolism , Frizzled Receptors/metabolism , Humans , Ligands , Models, Molecular , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Wnt Proteins/metabolism , beta Catenin/metabolismABSTRACT
Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.
Subject(s)
Cystine/metabolism , Peptides, Cyclic/metabolism , Protein Transport/physiology , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell-Penetrating Peptides/metabolism , Cyclization/physiology , Cyclotides/metabolism , Endocytosis/physiology , Endosomes/metabolism , HeLa Cells , Humans , Lysosomes/metabolism , Mice , NIH 3T3 Cells , Plant Proteins/metabolismABSTRACT
The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-ß-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and ß-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.
Subject(s)
Frizzled Receptors/metabolism , Protein Interaction Maps , Wnt Proteins/metabolism , Wnt Signaling Pathway , Animals , Cell Line , Mice , Phosphorylation , Protein Interaction Mapping , Protein Isoforms/metabolism , beta Catenin/metabolismABSTRACT
Cyclotides belong to the family of cyclic cystine-knot peptides and have shown promise as scaffolds for protein engineering and pharmacological modulation of cellular protein activity. Cyclotides are characterized by a cystine-knotted topology and a head-to-tail cyclic polypeptide backbone. While they are primarily produced in plants, cyclotides have also been obtained by chemical synthesis. However, there is still a need for methods to generate cyclotides in high yields to near homogeneity. Here, we report a biomimetic approach which utilizes an engineered version of the enzyme Sortase A to catalyze amide backbone cyclization of the recombinant cyclotide MCoTI-II, thereby allowing the efficient production of active homogenous species in high yields. Our results provide proof of concept for using engineered Sortase A to produce cyclic MCoTI-II and should be generally applicable to generating other cyclic cystine-knot peptides.
Subject(s)
Aminoacyltransferases/metabolism , Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , Cystine/chemistry , Peptides, Cyclic/chemistry , Peptides, Cyclic/metabolism , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Amino Acid Sequence , Aminoacyltransferases/chemistry , Aminoacyltransferases/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cyclization , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/genetics , Staphylococcus aureus/enzymologyABSTRACT
The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication/physiology , DNA, Fungal/biosynthesis , DNA-Binding Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , Minichromosome Maintenance Complex Component 3 , Minichromosome Maintenance Complex Component 4 , Minichromosome Maintenance Complex Component 6 , Minichromosome Maintenance Complex Component 7 , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding/physiology , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.
Subject(s)
Caspase 6/metabolism , Caspase Inhibitors/chemistry , Caspase Inhibitors/pharmacology , Amino Acid Sequence , Caspase 6/chemistry , Caspase Inhibitors/analysis , Crystallography, X-Ray , Drug Evaluation, Preclinical , Kinetics , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Protein Binding/drug effects , Reproducibility of Results , Substrate Specificity/drug effects , Surface Plasmon ResonanceABSTRACT
Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion of Caspase-6 indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other "upstream" caspases, prompting us to revisit the role of Caspase-3. In vitro, we show that genetic deletion of Caspase-3 is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from prosurvival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies. In vivo, Caspase-3 and Caspase-6-knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.
Subject(s)
Axons/enzymology , Caspase 3/physiology , Caspase 6/physiology , Nerve Degeneration/enzymology , Superior Colliculi/growth & development , Animals , Axons/pathology , Axons/ultrastructure , Caspase 3/genetics , Caspase 6/genetics , Cells, Cultured , Enzyme Activation/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Imaging/methods , Nerve Degeneration/genetics , Nerve Degeneration/pathology , Nerve Growth Factor/adverse effects , Proto-Oncogene Proteins c-bcl-2/physiology , Sensory Receptor Cells/enzymology , Sensory Receptor Cells/pathology , Signal Transduction/genetics , Signal Transduction/physiology , Superior Colliculi/enzymology , Wallerian Degeneration/enzymology , Wallerian Degeneration/genetics , Wallerian Degeneration/pathology , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/physiologyABSTRACT
The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.
Subject(s)
Biopolymers/metabolism , Caspase 6/metabolism , Enzyme Precursors/metabolism , Peptides/metabolism , Allosteric Regulation , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Humans , Peptides/chemistry , Protein BindingABSTRACT
Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Gene Expression Regulation, Fungal , Neurospora crassa/metabolism , Protein Phosphatase 2/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cell Wall/metabolism , MAP Kinase Signaling System , Molecular Sequence Data , Peptides/chemistry , Phosphoprotein Phosphatases/metabolism , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal Transduction , src Homology DomainsABSTRACT
The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of "negative" interactions in determining the specificity of a domain.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , src Homology Domains/physiology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Binding , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.