Workers of the ant Formica truncorum specialize in rearing females or males depending on the number of fathers of a colony. These split sex ratios increase inclusive fitness, but it has remained ...unknown how workers assess the number of patrilines in their colonies and to what extent their reproductive decisions are constrained by lack of information. By analysis of the quantitative variation in cuticular hydrocarbon profiles of workers of multiply mated queens, we show that the heritable component of recognition cues is low and that the extent of sex ratio biasing toward males is correlated with patriline differences in hydrocarbon profiles. Workers are thus able to capitalize on colony-level relatedness asymmetry, but their inclusive fitness is constrained by uninformative recognition cues. These results are consistent with the hypothesis that the occasional expression of nepotistic phenotypes favoring full-sisters over half-sisters maintains selection against informative recognition cues. We evaluate how inclusive fitness theory may be used to predict the number and kind of recognition cues in insect societies of a specific relatedness structure.
Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain ...elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl‐ACP to yield an extended β‐ketoacyl chain (ACP=acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans‐acyltransferase (trans‐AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.
Extension granted: In vitro studies on ketosynthase (KS) domains from trans‐acyltransferase polyketide synthases were carried out by varying the substrate (R group) and measuring product formation by mass spectrometry. The results revealed substrate selectivity for the chain elongation step that mirrors the predictions based on phylogenetic analysis. Substrate tolerance profiles provide valuable information for the bioengineering of polyketide assembly lines.
Glycosylation is often cited as having a stabilizing effect upon proteins with respect to proteolysis, thermolysis and other forms of degradation. We present here a model study on an autolytic ...protease that has been chemically glycosylated to produce single glycoforms. The resulting glycosylated enzymes are more stable with respect to their own autolytic degradation and that by other proteases. Kinetic parameters for protease activity with respect to the degradation of small-molecule amide substrate reveal no significant change in inherent activity thereby suggesting that reduced autolysis and proteolysis are a consequence of stabilization, perhaps by steric blockade of cleavage points or alteration of local unfolding kinetics. Variation in glycan identity suggests that greater glycan size leads to greater stabilization.
Conforming to convention: Evidence for compact and extended forms of cytochrome P450 reductase, believed to exist in dynamic equilibrium in solution, is found in the gas phase by using ion mobility ...spectrometry. The relative abundance of the two conformations (see picture) can be influenced by the ionic strength of the solution from which they are electrosprayed.
Site‐selective glycosylation by SeS‐mediated ligation has led to the efficient formation of a wide variety of conjugates 1 without the need for a large excess of the carbohydrate reagent. By this ...convergent method it was possible to introduce a heptasaccharide glycan selectively, and to perform a multiple site‐selective chemical glycosylation of protein. A chemically Cys‐glycosylated glycoprotein was elaborated enzymatically.
Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through ...recognition of the 5′-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-(5,6-dihydro-6-oxo-1,3-dioxolo4,5-gquinolin-7-yl)methyl-N′-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5′-deoxy-5′-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5′-deoxy-5′-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5′-deoxy-5′-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents.
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Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding ...interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 (C285A) active-site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter, consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type hCaspase-1, VRT-043198 modifies the active-site Cys285 through the formation of a S,O-hemiacetal. Here, we showed by carbene labeling that this inhibitor can noncovalently occupy the active site of a C285A mutant. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system.
Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate‐conjugated polyubiquitin. However tools able to ...discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnF‐UBP domain in tandem with a linkage‐selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48‐linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48‐linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t‐UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48‐linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain‐selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the ‘ubiquitome’. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).
The trans-acyltransferase (AT) polyketide synthases are a recently recognised group of bacterial enzymes that generate complex polyketides. A prerequisite for re-engineering these poorly studied ...systems is knowledge about the substrate specificity of their components. In this work, KS domain 1 from the bacillaene polyketide synthase has been shown to possess high specificity towards 2-amidoacetyl intermediates, which are derived from incorporation of alpha amino acids into the polyketide chain. N-Acetylcysteamine (SNAC) analogues of full-length substrates were synthesised and incubated with the KS1 domain. The natural glycine-derived acyl-SNAC was found to acylate KS1 with highest efficiency, as evidenced by mass spectrometry (MS). An alanine variant was also incorporated, but its valine equivalent was not, which indicated limited tolerance of substitution at the alpha -position. Substrate analogues without an amine or amide nitrogen substituted on the 2-position were not accepted by KS1 at the standard assay concentration of 0.5 mM. Moreover, removal of Asn-206 from the active site of KS1 by site-directed mutagenesis reduced k sub(cat)/K sub(m) by a factor of approx. 2. This residue is conserved in most known 2-amidoacetyl-accepting KS domains from trans-AT PKSs and we postulate an important interaction between Asn-206 and the amide nitrogen of the substrate.
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