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 pro-survival 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.
Background: WNT-FZD specificity and signaling outcome have not been systematically mapped so far. Results: WNTs show selective binding to FZDs, and respective WNT-FZD pairs exert functional selectivity in different downstream signaling pathways. Conclusion: The WNT-FZD signaling system provides ligand-receptor selectivity. Significance: Understanding WNT-FZD selectivity is crucial for development of WNT pathway inhibitors and further understanding of molecular interactions within the WNT receptor signaling complex.
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)...
Edited by Miguel De la RosaKeywords: Cyclotide Recombinant expression Sortase Protein engineering MCoTI-II Cystine-knot peptide a b s t r a c t 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.
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.
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.The ubiquitin pathway is conserved throughout eukaryotic evolution and is implicated in numerous cellular processes (1). Proteins modified by the ubiquitin pathway are processed for degradation, endocytosis, protein sorting, and subnuclear trafficking (2, 3). Ubiquitination is catalyzed by three enzymes termed E1 1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin protein ligase). E3 regulates the specificity of the reaction by binding directly to substrates (1, 3). The E3-substrate interaction is implicated in an increasing number of diseases, including neurodegeneration, immunological disorders, hypertension, and cancers. For example, numerous E3 enzymes such as Fbw7, Skp2, Mdm2, and VHL and their respective substrates, cyclin E, p27, p53, and HIF, have been linked to tumor progression (4, 5). The therapeutic importance of understanding ubiquitination has been underscored recently by the success of anticancer strategies that affect the ubiquitin pathway (6).Most, if not all, proteins are regulated by the ubiquitin pathway. A recent proteomic approach identified over a thousand proteins that are ubiquitinated in yeast under normal conditions (7). This study, which likely did not detect many nonabundant proteins or proteins that are ubiquitinated under specific conditions (e.g. stress and nutrition), underlines the breadth of the ubiquitin system. Current estimates also predict that there are hundreds of E3 enzymes in eukaryotic genomes (8) whose role is to ubiquitinate these proteins. Despite the biomedical importance of E3 enzymes and great advances in understanding the mechanics of the ubiquitin system, a very small fraction of E3 enzymes has been linked to specific substrates, and currently, the scarcity of identified E3-substrate pairs in the literature is a major bottleneck in the ubiquitin field.Rsp5 is a yeast E3 enzyme, and many of its substrates have not yet been characterized. It belongs to the Nedd4 family of E3 ligase...
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.
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