Dissecting the Oligomeric Behavior of Caveolin-1 using the Analytical Ultracentrifuge

https://nsuworks.nova.edu/nsudigital_harrison/3450/thumbnail.jp

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Investigating Detergent and Lipid Systems for the Study of Membrane Protein Interactions: Characterizing Caveolin Oligomerization

Membrane proteins represent an important class of proteins that closely associate or reside within the plasma membrane of the cell. They play a multitude of roles in cell function such as signaling, trafficking, and recently discovered, scaffolding and shaping of the plasma membrane itself. For example, caveolin is a membrane protein that is believed to have the ability to curve the plasma membrane forming invaginations that serve as signaling platforms called caveolae. The curvature of the plasma membrane is believed to be a result of caveolin oligomerization. Caveolin oligomerization was characterized using sedimentation equilibrium analytical ultracentrifugation. Due to the extremely hydrophobic nature of caveolin it was necessary to explore different detergents and lipid systems that support membrane protein structure and function. Not all detergents are conducive to studies of membrane proteins and it is often necessary to determine empirically the best detergent / lipid mimic best suited for biophysical studies. One membrane mimic that has been well-characterized and used successfully to study membrane proteins are bicelles. Bicelles are discoidal phospholipid structures comprised of a long-chain and short-chain phospholipid, typically 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC), respectively. Bicelles provide a true bilayer environment in which to study membrane protein structure and function. These lipid structures were successfully density matched using the method of sedimentation equilibrium in the analytical ultracentrifuge by adding 71.7% D2O as a density modifier. We explored the utility of bicelles as a medium for studying membrane protein interactions in the analytical ultracentrifuge (AUC) by investigating the interactions of caveolin-1. The results of this work show that caveolin-1 does not have the capacity to oligomerize in detergent micelles or in a bilayer environment (bicelles). On the other hand, a naturally-occuring breast cancer mutant, P132L, forms a strong dimer in detergent micelles. A close investigation of the mutant reveals that an extension of helix 2 in the intramembrane region of the protein where dimerization was shown to occur may play a key role in the dimerization of the mutant. An alternative bicelle system was also investigated using pentaethylene glycol monooctyl ether (C8E5) instead of DHPC to form the rim of the bicelle. The C8E5 / DMPC lipid aggregates were density matched and their properties were characterized using 31P-phosphorus NMR to assess the heterogeneity of the lipid / detergent arrangement, which confirms a bicellar-like arrangement. C8E 5 has a density similar to water (1.007 g / mL) and was shown to form lipid aggregate structures with DMPC that are less dense and require significantly lower quantity of D2O to density match in the AUC making them better suited to the study of membrane protein interactions of small peptides

Adenosine A2a receptors form distinct oligomers in protein detergent complexes

The human adenosine A2a receptor (A2aR) tunes its function by forming homo-oligomers and hetero-oligomers with other GPCRs, but the biophysical characterization of these oligomeric species is limited. Here, we show that upon reconstitution into an optimized mixed micelle system, and purification via an antagonist affinity column, full length A2aR exists as a distribution of oligomers. We isolated the dimer population from the other oligomers via size exclusion chromatography and showed that it is stable upon dilution, thus supporting the hypotheses that the A2aR dimer has a defined structure and function. This study presents a crucial enabling step to a detailed biophysical characterization of A2aR homodimers

Probing the Caveolin-1 P132L Mutant: Critical Insights into Its Oligomeric Behavior and Structure

Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation <i>in vivo</i> and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation, we found that a fully functional construct of caveolin-1 (Cav1_62–178) was a monomer in dodecylphosphocholine micelles. In contrast, the P132L mutant of Cav1_62–178 was dimeric. To explore the dimerization of the P132L mutant further, various truncated constructs (Cav1_82–178, Cav1_96–178, Cav1_62–136, Cav1_82–136, Cav1_96–136) were prepared which revealed that oligomerization occurs in the transmembrane domain (residues 96–136) of caveolin-1. To characterize the mutant structurally, solution-state NMR experiments in <i>lyso</i>-myristoylphosphatidylglycerol were undertaken of the Cav1_96–136 P132L mutant. Chemical shift analysis revealed that, compared to the wild-type, helix 2 in the transmembrane domain was lengthened by four residues (wild-type, residues 111–129; mutant, residues 111–133), which corresponds to an extra turn in helix 2 of the mutant. Lastly, point mutations at position 132 of Cav1_62–178 (P132A, P132I, P132V, P132G, P132W, P132F) revealed that no other hydrophobic amino acid can preserve the monomeric state of Cav1_62–178, which indicates that proline 132 is critical in supporting proper caveolin-1 behavior