The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in ...designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets—called hidden allosteric sites—remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond- to millisecond-timescale fluctuations of a protein’s structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target—TEM-1 β-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.
Significance Rational drug design efforts typically focus on identifying inhibitors that bind to protein active sites. Pockets that are not present in crystallographic structures yet can exert allosteric (i.e., long-range) control over distant active sites present an exciting alternative. However, identifying these hidden allosteric sites is extremely challenging because one usually has to simultaneously find a small molecule that binds to and stabilizes the open conformation of the pocket. Here, we present a means of combining advances in computer modeling—using Markov state models to capture long timescale dynamics—with biophysical experiments to identify hidden allosteric sites without requiring the simultaneous discovery of drug-like compounds that bind them. Using this technology, we discover multiple hidden allosteric sites in a single protein.
Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO
-concentrating mechanism by facilitating high CO
concentrations near the carboxylase Rubisco. The ...carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.
•Fetal magnetocardiography was acquired with a semi-rigid prototype optically-pumped magnetometers (OPM) sensor array.•OPM system has the ability to record quality fetal cardiac activities and is ...capable of identifying lethal cardiac rhythm disturbances in the fetus across gestation ages.•We were able to quantify the variation in fetal heart rate viability parameters.•Fetal biomagnetometry with OPM could be translated in the future from research to widespread clinical applications.
To record and extract features of fetal cardiac activities with a semi-rigid prototype optically-pumped magnetometers (OPM) sensor array.
Fetal magnetocardiography (fMCG) data were collected from 15 pregnant women between 28 and 40 weeks gestation. Mothers were lying flat in a customized bed with sensors touching their abdomen from below using a prototype grid. fMCG was extracted to perform standard fetal heart rate variability (FHRV) analysis.
fMCG was observed in 13 of the 15 pregnant women. OPM FHRV indicators were in the range of previous SQUID studies.
Semi-rigid prototype OPM system has the ability to record quality fMCG. fMCG is capable of identifying lethal cardiac rhythm disturbances in the fetus. Our novel application of OPM technology may lower costs and increase maternal comfort, thus expanding fMCG's generalizability.
Key points
What's already known about this topic?
A disproportionate number of children with cyanotic heart disease suffer from neurodevelopmental (ND) impairment, which is believed to be due in part ...to abnormal cerebral development in utero. Magnetoencephelography (MEG) is a non‐invasive method for assessing neurodevelopment, but has not been studied in fetuses with congenital heart disease (CHD).
What does this study add?
In this preliminary study, we showed the feasibility of performing MEG in fetuses with CHD. Fetuses with CHD had different magnetoencephalographic characteristics when compared with matched controls.
Fetal sex has been associated with different development trajectories that cause structural and functional differences between the sexes throughout gestation. Fetal magnetocardiography (fMCG) ...recordings from 123 participants (64 females and 59 males; one recording/participant) from a database consisting of low-risk pregnant women were analyzed to explore and compare fetal development trajectories of both sexes. The gestational age of the recordings ranged from 28 to 38 weeks. Linear metrics in both the time and frequency domains were applied to study fetal heart rate variability (fHRV) measures that reveal the dynamics of short- and long-term variability. Rates of linear change with GA in these metrics were analyzed using general linear model regressions with assessments for significantly different variances and GA regression slopes between the sexes. The fetal sexes were well balanced for GA and sleep state. None of the fHRV measures analyzed exhibited significant variance heterogeneity between the sexes, and none of them exhibited a significant sex-by-GA interaction. The absence of a statistically significant sex-by-GA interaction on all parameters resulted in none of the regression slope estimates being significantly different between the sexes. With high-precision fMCG recordings, we were able to explore the variation in fHRV parameters as it relates to fetal sex. The fMCG-based fHRV parameters did not show any significant difference in rates of change with gestational age between sexes. This study provides a framework for understanding normal development of the fetal autonomic nervous system, especially in the context of fetal sex.
Objective: Fetal magnetocardiography (fMCG) is a non-invasive biomagnetic technique that provides detailed beat-to-beat fetal heart rate analysis, both in normal rhythm as well as in fetal ...arrhythmias. New cryogenic-free sensors called optically pumped magnetometers (OPMs) have emerged as a less expensive and more geometrically flexible alternative to traditional Superconducting Quantum Interference Device (SQUID) technology for performing fMCG. The objective of the study was to show the ability of OPMs to record fMCG using flexible geometry while seeking to preserve signal quality, and to quantify fetal heart rate variability (FHRV). Approach: Biomagnetic measurements were performed with OPMs in 24 healthy pregnant women with uncomplicated singleton pregnancies between 28 and 38 weeks gestation (GA). A total of 96 recordings were analyzed from OPM data that was collected using sensors placed in two different maternal configurations over the abdomen. The fMCG signals were extracted and the quality of the recordings were quantified by peak amplitudes and signal-to-noise ratio (SNR). R peaks were used to perform both time and frequency domain FHRV analysis. FHRV measures obtained from OPMs were compared descriptively to the same measures obtained from GA-matched existing SQUID data. Main results: The fMCG derived from OPMs were observed in 21 of the 24 participants. Higher detection rates (85%) of fMCG signals were observed in the data sets recorded at GA >32 weeks. Peak amplitudes and SNR values were similar between two maternal configurations, but peak amplitudes were significantly higher (p = 0.013) in late GA compared to early GA. FHRV indicators were successfully extracted and their values overlapped substantially with those obtained from SQUID recordings. Significance: Taking advantage of the geometric flexibility of the OPMs, we have demonstrated their ability to record and quantify fMCG in different maternal positions as opposed to rigid SQUID configurations.
Aim
Pregnant women undergoing treatment for opioid use disorder (OUD) may be exposed to multiple QT prolonging agents. We used magnetocardiography to measure fetal QT intervals in mothers with OUD on ...buprenorphine therapy.
Methods
Fetal and maternal magnetocardiography was performed in pregnant women receiving buprenorphine‐assisted treatment (Disorder group); these were matched by gestational age to pregnant women who were opiate naïve (Reference group). Corrected QT intervals were determined using Bazett's formula and compared between groups.
Results
A total of eight women in the Disorder group matched to eight in the Reference group. Seven of the mothers (88%) in the Disorder group were smokers; there were no smokers in the Reference group. The average fetal corrected QT was significantly longer (P = 0.022) in the Disorder group than that in the Reference group (505 milliseconds ms ± 68.6 standard deviation vs 383 ms ± 70.3 standard deviation).
Conclusion
Novel data from this small sample demonstrate prolongation of fetal corrected QT in women with OUD participating in buprenorphine assisted therapy. Additional investigation from a larger sample is needed to clarify if fetal buprenorphine and/or tobacco exposure is associated with changes in fetal QT which would warrant further prenatal and postnatal testing.
Zinc (Zn) deficiency and obesity are global public health problems. Zn deficiency is associated with obesity and comorbid conditions that include insulin resistance and type 2 diabetes. However, the ...function of Zn in obesity remains unclear. Using a mouse model of combined high-fat and low-Zn intake (0.5-1.5 mg/kg), we investigated whether Zn deficiency exacerbates the extent of adiposity as well as perturbations in metabolic and immune function. C57BL/6 mice were randomly assigned to receive either a high-fat diet (HFD) or a control (C) diet for 6 wk, followed by further subdivision into 2 additional groups fed Zn-deficient diets (C-Zn, HFD-Zn), along with a C diet and an HFD, for 3 wk (n = 8-9 mice/group). The extent of visceral fat, insulin resistance, or systemic inflammation was unaffected by Zn deficiency. Strikingly, Zn deficiency significantly augmented circulating leptin concentrations (HFD-Zn vs. HFD: 3.15 ± 0.16 vs. 2.59 ± 0.12 μg/L, respectively) and leptin signaling in the liver of obese mice. Furthermore, gene expression of macrophage-specific markers ADAM8 (A disintegrin and metalloproteinase domain-containing protein 8) and CD68 (cluster of differentiation 68) was significantly greater in adipose tissue in the HFD-Zn group than in the HFD group, as confirmed by CD68 protein analysis, indicative of increased macrophage infiltration. Inspection of Zn content and mRNA profiles of all Zn transporters in the adipose tissue revealed alterations of Zn metabolism to obesity and Zn deficiency. Our results demonstrate that Zn deficiency increases leptin production and exacerbates macrophage infiltration into adipose tissue in obese mice, indicating the importance of Zn in metabolic and immune dysregulation in obesity.
1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly ...N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolι), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dGAP on a synthetic DNA template but that hRev1 was stopped after lesion bypass. The time required to bypass 50% of the dGAP sites (t 50 bypass) encountered by hPolη, hPolκ, and hPolι was determined to be 2.5 s, 4.1 s, and 106.5 s, respectively. The efficiency order of catalyzing translesion synthesis of dGAP (hPolη > hPolκ > hPolι ≫ hRev1) is the same as the order for these human Y-family enzymes to elongate undamaged DNA. Although hPolη bypassed dGAP efficiently, replication by both hPolκ and hPolι was strongly stalled at the lesion site and at a site immediately downstream from dGAP. By employing presteady state kinetic methods, a kinetic basis was established for polymerase pausing at these DNA template sites. Besides efficiency of bypass, the fidelity of those low-fidelity polymerases at these pause sites was also significantly decreased. Thus, if the translesion DNA synthesis of dGAP in vivo is catalyzed by a human Y-family DNA polymerase, e.g., hPolη, the process is certainly mutagenic.
Zinc (Zn) deficiency and obesity are global public health problems. Zn deficiency is associated with obesity and comorbid conditions that include insulin resistance and type 2 diabetes. However, the ...function of Zn in obesity remains unclear. Using a mouse model of combined high-fat and low-Zn intake (0.5–1.5 mg/kg), we investigated whether Zn deficiency exacerbates the extent of adiposity as well as perturbations in metabolic and immune function. C57BL/6 mice were randomly assigned to receive either a high-fat diet (HFD) or a control (C) diet for 6 wk, followed by further subdivision into 2 additional groups fed Zn-deficient diets (C-Zn, HFD-Zn), along with a C diet and an HFD, for 3 wk (n = 8–9 mice/group). The extent of visceral fat, insulin resistance, or systemic inflammation was unaffected by Zn deficiency. Strikingly, Zn deficiency significantly augmented circulating leptin concentrations (HFD-Zn vs. HFD: 3.15 ± 0.16 vs. 2.59 ± 0.12 μg/L, respectively) and leptin signaling in the liver of obese mice. Furthermore, gene expression of macrophage-specific markers ADAM8 (A disintegrin and metalloproteinase domain-containing protein 8) and CD68 (cluster of differentiation 68) was significantly greater in adipose tissue in the HFD-Zn group than in the HFD group, as confirmed by CD68 protein analysis, indicative of increased macrophage infiltration. Inspection of Zn content and mRNA profiles of all Zn transporters in the adipose tissue revealed alterations of Zn metabolism to obesity and Zn deficiency. Our results demonstrate that Zn deficiency increases leptin production and exacerbates macrophage infiltration into adipose tissue in obese mice, indicating the importance of Zn in metabolic and immune dysregulation in obesity.
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