Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with k max/K M = 2.1 × 103 and 3.7 × 103 M–1 s–1, respectively, and an identical pK a = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.
Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔG°) of -4.4 and -6.9 kcal mol for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔV) was significantly larger for V67 (71 ± 2 mL mol) than for L67 (58 ± 3 mL mol) inteins. The measured difference in ΔV (13 ± 3 mL mol) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.
Background Obesity has become a prevalent public health issue, increasing the risk of cardiovascular diseases which are the leading cause of death in the US. A major contributor to the obesity epidemic is the chronic consumption of diets rich in saturated fat and sugar, known as western diet (WD). During obesity, PVAT loses its vasculo‐protective properties and becomes inflamed negatively impacting vascular function. Cx3cr1GFP/WT transgenic fluorescent mice that express a GFP reporter in microglia/macrophages, were utilized in this study to visualize macrophage infiltration in the PVAT from mice chronically exposed to a WD. Methods Adult homozygous male Cx3cr1GFP/WT mice were randomized to the control group (n=4) fed a regular chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], and 24.1% protein) and the WD group (n=9) fed a WD (40% fat, 43% carbohydrates [34% sucrose], and 17% protein) for 52 weeks. Metabolic cage studies were performed to determine food and water intake, along with feces and urine output. Glucose metabolism was assessed by intraperitoneal glucose tolerance test (IPGTT). At the terminal experiments, direct measurements of arterial blood pressure were obtained by carotid catheterization, and aortas containing PVAT were collected for further histological analysis. Macrophage labeled by GFP in PVAT was visible by fluorescence microscopy and scored in a blind fashion. Results As expected, after over one year under WD conditions, Cx3cr1GFP/WT mice exhibited increased body weight (40.82 ± 2.09 vs. 33.83 ± 1.77g controls, p<0.05) and intolerance to glucose as demonstrated by increased blood glucose area under the curve during IPGTT (51828 ± 4562 vs. 28333 ± 3182 a.u, p<0.05). While the WD group showed decreased daily food consumption (1.36 ± 0.24 vs. 2.17 ± 0.10g controls, p<0.05), no differences in caloric consumption was observed between the groups (5.56 ± 2.78 vs 5.36 ± 1.37 kcals controls). Interestingly, the WD group exhibited decreased water intake (2.90 ± 0.92 vs. 5.13 ± 1.96 mL, p<0.05), urine output (0.56 ± 0.32 vs.1.69 ± 0.63 ml, p<0.01), and feces output (0.34 ± 0.10 vs.1.66 ± 0.30g, p<0.0001) compared to controls. Chronic exposure to WD also resulted in increased systolic blood pressure (119.1 ± 5 vs. 102.9 ± 4 mmHg, p<0.05) along with increased heart rate (481.2 ± 11.7 vs. 429.1 ± 24.6 bpm, p<0.05). Interestingly, PVAT from the WD group exhibited a white‐like adipose tissue feature comprising enlarged adipocytes, in contrast to their native brown‐like adipose tissue, characterizing a phenotypic modulation. Strikingly, GFP signal in the PVAT from the WD group was significantly augmented by 70% compared to controls (Figure 1), showing a robust infiltration of macrophages in the PVAT. Conclusion Our results suggest that increased infiltration of macrophages in PVAT after chronic consumption of a WD promotes inflamed PVAT which in turn may contribute to dysregulation of blood pressure. Further studies will be needed to fully characterize the negative impact of a WD in the PVAT and its cons...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.