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Bound cholesterol molecules are emerging as important hallmarks of GPCR structures. In this commentary, we analyze their statistical prevalence and biological relevance.Graphic Abstract

The dynamic interactions between G protein-coupled receptors (GPCRs) and their cognate protein partners are central to several cell signaling pathways. For example, the association of CXC chemokine receptor 1 (CXCR1) with its cognate chemokine, interleukin-8 (IL8 or CXCL8) initiates pathways leading to neutrophil-mediated immune responses. The N-terminal domain of chemokine receptors confers ligand selectivity, but unfortunately the conformational dynamics of this intrinsically disordered region remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations, complemented by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo -receptor N-terminal domain is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we corroborated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies and show that the trends are similar. Our results indicate that chemical shift perturbation is often not a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.

The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer's disease, that are associated with impaired cholesterol biosynthesis and homeostasis.

Cholesterol plays a crucial role in cell membrane organization, dynamics and function. Depletion of cholesterol represents a popular approach to explore cholesterol-sensitivity of membrane proteins. An emerging body of literature shows that the consequence of membrane cholesterol depletion often depends on the actual process (acute or chronic), although the molecular mechanism underlying the difference is not clear. Acute depletion, using cyclodextrin-type carriers, is faster relative to chronic depletion, in which inhibitors of cholesterol biosynthesis are used. With the overall goal of addressing molecular differences underlying these processes, we monitored membrane dipole potential under conditions of acute and chronic cholesterol depletion in CHO-K1 cells, using a voltage-sensitive fluorescent dye in dual wavelength ratiometric mode. Our results show that the observed membrane dipole potential exhibits difference under acute and chronic cholesterol depletion conditions, even when cholesterol content was identical . To the best of our knowledge, these results provide, for the first time, molecular insight highlighting differences in dipolar reorganization in these processes. A comprehensive understanding of processes in which membrane cholesterol gets modulated would provide novel insight in its interaction with membrane proteins and receptors, thereby allowing us to understand the role of cholesterol in cellular physiology associated with health and disease.

The cell cycle is a sequential multi-step process essential for growth and proliferation of cells comprising multicellular organisms. Although a number of proteins are known to modulate the cell cycle, the role of lipids in regulation of cell cycle is still emerging. In our previous work, we monitored the role of cholesterol in cell cycle progression in CHO-K1 cells. Since sphingolipids enjoy a functionally synergistic relationship with membrane cholesterol, in this work, we explored whether sphingolipids could modulate the eukaryotic cell cycle using CHO-K1 cells. Sphingolipids are essential components of eukaryotic cell membranes and are involved in a number of important cellular functions. To comprehensively monitor the role of sphingolipids on cell cycle progression, we carried out metabolic depletion of sphingolipids in CHO-K1 cells using inhibitors (fumonisin B1, myriocin, and PDMP) that block specific steps of the sphingolipid biosynthetic pathway and examined their effect on individual cell cycle phases. Our results show that metabolic inhibitors led to significant reduction in specific sphingolipids, yet such inhibition in sphingolipid biosynthesis did not show any effect on cell cycle progression in CHO-K1 cells. We speculate that any role of sphingolipids on cell cycle progression could be context and cell-type dependent, and cancer cells could be a better choice for monitoring such regulation, since sphingolipids are differentially modulated in these cells.Graphic Abstract

ObjectivesNeurodevelopmental disability is the most common long-term consequence for survivors of surgery for congenital heart disease (CHD). The primary objectives of this prospective multi-centre hospital-based cohort study were to determine the prevalence and predictors of motor and mental neurodevelopmental delay using the developmental assessment scale for Indian infants (DASII) at twelve months after corrective cardiac surgery using bypass from five centres across India.MethodsThe estimated sample size was 1250 based on Gaynor et al.1 Participants were recruited from five tertiary cardiac care centres across India – Delhi, Mumbai, Kolkata, Bangalore and Kochi. Neuro-developmental assessment was done in hospital using DASII. All assessments were conducted at study institutes by trained Neuro-developmental assessors. We reported the prevalence of motor as well as mental neurodevelopmental delay in general as well as stratified for 14 clusters as per DASII. We used multivariate logistic regression analysis to report adjusted Odds Ratios separately for predictors of motor and mental developmental delay.ResultsWe recruited 1753 infants. A total of 1292 infants (73.7%) were eligible for follow up assessment at twelve months after cardiac surgery. A total of 111 infants (6.3%) expired in hospital and another 77 (4.4%) expired during one year follow up after discharge. A total of 299 participants (24.6%) were lost to follow up. We analysed data from remaining 916 infants. A total of 131 infants (14.3%) had motor developmental delay and 94 (10.3%) had mental developmental delay. In cluster-based analysis, maximum delay was seen for body control among motor clusters (n 285, 31.1%). Manual dexterity showed maximum delay (n 319, 34.8%) among mental clusters. Significant predictors for motor delay included syndromic association (OR 28.6., 95% CI 14.1, 58.0) and duration of mechanical ventilation in hours (OR 1.003, 95% CI 1.001 – 1.005). The corresponding predictors for mental delay were syndromic association (OR 44.8, 95% CI 21.8, 92.1), pre-operative sepsis (OR 3.2, 95% CI 1.6, 6.3), birth weight (OR 0.5, 95% CI 0.295, 0.845), duration of mechanical ventilation in hours (OR 1.004, 95% CI 1.002, 1.006) and highest lactate in ICU stay(OR 1.103, 95%CI 1.007, 1.208).ConclusionsApproximately one in six among survivors of CHD surgery during infancy experience neurodevelopmental morbidity at one year after cardiac surgery. Significant predictors of neurodevelopmental delay include syndromic association, pre-operative sepsis, duration of mechanical ventilation, birth weight and highest lactate during ICU stay. Cluster based developmental assessment shows deficiencies in multiple sub domains highlighting the need for detailed neurodevelopmental assessment among survivors of CHD during post-surgical follow up. There is a steady increase in the number of high risk infants with CHD who undergo corrective cardiac surgery and survive to adulthood. This ever increasing population of survivors will require significant societal resources for appropriate neurodevelopmental assessment, regular follow up and necessary interventions.

ObjectivesNeurodevelopmental disability is the most common long-term consequence for survivors of surgery for congenital heart disease (CHD). The primary objectives of this prospective multi-centre hospital-based cohort study were to determine the prevalence and predictors of motor and mental neurodevelopmental delay using the developmental assessment scale for Indian infants (DASII) at six months after corrective cardiac surgery using bypass from five centres across India.MethodsWe followed up 1143 infants who were operated for CHD in the first year of life. Infants were recruited from five tertiary cardiac care centres across India – Delhi, Mumbai, Kolkata, Bangalore and Kochi. Neuro-developmental assessment was done in hospital using DASII in a designated neurodevelopmental assessment room by trained Neuro-developmental assessors recruited for this study. We reported the prevalence of motor as well as mental neurodevelopmental delay in general as well as stratified for 14 clusters as per DASII. We did logistic regression to report the adjusted Odds Ratios for predictors of overall motor and mental developmental delay.ResultsThe estimated sample size was 1250 based on Gaynor et al.1 We recruited 1143 infants. A total of 995 infants (87%) were alive at six months after cardiac surgery. A total of 92 infants (8.0%) expired in hospital and another 56 (4.9%) expired during follow up after discharge. We analysed data from remaining 816 infants. A total of 234 infants (28.7%) had motor developmental delay and 143 (17.5%) had mental developmental delay. In cluster-based analysis, maximum delay was seen for body control among motor clusters (n 455, 55.8%). Social interaction and imitative behaviour showed maximum delay (n 382, 46.8%) among mental clusters. Significant predictors for motor delay included syndromic association (OR 11.8, 95% CI 5.3, 26.5), preoperative condition as measured by PRACHS score (OR 1.2, 95% CI 1.04,1.4) and duration of mechanical ventilation in hours (OR 1.003, 95% CI 1.001 – 1.004). The corresponding predictors for mental delay were syndromic association (OR 19.3, 95% CI 9.3, 39.9), post-operative sepsis (OR 2.0, 95% CI 1.1, 3.7) and duration of mechanical ventilation in hours (OR 1.003, 95% CI 1.002, 1.005).ConclusionA significant proportion of survivors of CHD surgery during infancy experience neurodevelopmental morbidity. Significant predictors of neurodevelopmental delay include syndromic association, preoperative condition, post-operative sepsis and duration of mechanical ventilation. Cluster based developmental assessment shows delays in multiple sub-domains. As more high-risk CHD infants undergo cardiac surgery and survive in low resource settings like India, a growing population will require significant societal resources.ReferenceGaynor JW, Stopp C, Wypij D, Andropoulos DB, Atallah J, Atz AM, Beca J, Donofrio MT, Duncan K, Ghanayem NS, Goldberg CS, Hövels-Gürich H, Ichida F, Jacobs JP, Justo R, Latal B, Li JS, Mahle WT, McQuillen PS, Menon SC, Pemberton VL, Pike NA, Pizarro C, Shekerdemian LS, Synnes A, Williams I, Bellinger DC, Newburger JW, International Cardiac Collaborative on Neurodevelopment (ICCON) Investigators. Neurodevelopmental outcomes after cardiac surgery in infancy. Pediatrics. 2015 May;135(5):816–25.

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. The organization of membrane-bound melittin has earlier been shown to be dependent on the physical state and composition of membranes. In this study, we covalently labeled the N-terminal (Gly-1) and Lys-7 of melittin with an environment-sensitive fluorescent probe, the NBD group, to monitor the influence of negatively charged lipids and cholesterol on the organization and dynamics of membrane-bound melittin. Our results show that the NBD group of melittin labeled at its N-terminal end does not exhibit red edge excitation shift in DOPC and DOPC/DOPG membranes, whereas the NBD group of melittin labeled at Lys-7 exhibits REES of ∼8 nm. This could be attributed to difference in membrane microenvironment experienced by the NBD groups in these analogs. Interestingly, the membrane environment of the NBD groups is sensitive to the presence of cholesterol, which is supported by time-resolved fluorescence measurements. Importantly, the orientation of melittin is found to be parallel to the membrane surface as determined by membrane penetration depth analysis using the parallax method in all cases. Our results constitute the first report to our knowledge describing the orientation of melittin in cholesterol-containing membranes. These results assume significance in the overall context of the role of membrane lipids in the orientation and function of membrane proteins and peptides.