Clinical severity and high-resolution CT severity score in COVID-19: Is there an association.

Objectives: To identify a correlation between the clinical parameters and CT chest severity score in COVID-19. Methods: A total of 205 RT-PCR positive patients were included in this descriptive cross-sectional study with convenience sampling from November 2020 to June 2021 in KRL Hospital. The study population was stratified in disease severity as per the WHO's guidelines. Clinical and radiological characteristics were compared in survivors and non survivors to draw conclusion. Results: The mean age was 57 years and the majority of the patients 57% were male. Overall mortality was 22% and the mean CT severity score was 18. Non survivors were more tachypneic, hypoxic, had a higher CT chest severity score, higher clinical severity, more comorbid condition and higher TLC, D-Dimers, LDH, CRP, NLR. Raised CT severity score showed a conclusive correlation with greater disease severity. One way ANOVA showed a significant difference between mean CT severity score amongst different disease categories. Conclusion: Higher CT severity score corresponds to a higher clinical severity and higher chances of mortality.

Predictors of Mortality in COVID-19 patients: An observational study.

Objectives: To identify the factors that affect outcome in COVID-19 patients in the Pakistani population. Methods: A total of 225 patients of COVID-19 RT-PCR proven were included during November, 2020 to June, 2021 in this cross-sectional study. They were stratified into different disease severity categories as per WHO guidelines. The characteristics of survivors and non survivors were recorded and then compared to draw conclusions. Results: Mean age was 59 years. Majority of the patients were male (68%) and the overall mortality rate was 30.1%. The non survivors were more likely to be female, had a greater number of comorbidities, had a higher respiratory rate and lower oxygen saturations at presentation and had a greater frequency of invasive mechanical ventilation. Non survivors had higher values of TLC, CRP, D-dimers and lower values of Hemoglobin and Platelets. The non survivors had higher incidence of ARDS, Septic shock and Multiorgan involvement. A higher CURB-65 score was observed in non survivors as compared to those who survived. Multivariate analysis showed that female gender, presence of and higher number of comorbid conditions and a higher CURB-65 score was linked with mortality. Conclusion: Results are compatible with international studies; increasing age, number of comorbid conditions and high inflammatory markers are associated with increased mortality. Our study had an exception that female gender had higher mortality as compared to men.

Mechanistic Insights into Protein Stability and Self-aggregation in GLUT1 Genetic Variants Causing GLUT1-Deficiency Syndrome.

Human sodium-independent glucose cotransporter 1 (hGLUT1) has been studied for its tetramerization and multimerization at the cell surface. Homozygous or compound heterozygous mutations in hGLUT1 elicit GLUT1-deficiency syndrome (GLUT1-DS), a metabolic disorder, which results in impaired glucose transport into the brain. The reduced cell surface expression or loss of function have been shown for some GLUT1 mutants. However, the mechanism by which deleterious mutations affect protein structure, conformational stability and GLUT1 oligomerization is not known and require investigation. In this review, we combined previous knowledge of GLUT1 mutations with hGLUT1 crystal structure to analyze native interactions and several natural single-point mutations. The modeling of native hGLUT1 structure confirmed the roles of native residues in forming a range of side-chain interactions. Interestingly, the modeled mutants pointed to the formation of a variety of non-native novel interactions, altering interaction networks and potentially eliciting protein misfolding. Self-aggregation of the last part of hGLUT1 was predicted using protein aggregation prediction tool. Furthermore, an increase in aggregation potential in the aggregation-prone regions was estimated for several mutants suggesting increased aggregation of misfolded protein. Protein stability change analysis predicted that GLUT1 mutant proteins are unstable. Combining GLUT1 oligomerization behavior with our modeling, aggregation prediction, and protein stability analyses, this work provides state-of-the-art view of GLUT1 genetic mutations that could destabilize native interactions, generate novel interactions, trigger protein misfolding, and enhance protein aggregation in a disease state.

Identification of phlorizin binding domains in sodium-glucose cotransporter family: SGLT1 as a unique model system.

The sodium glucose cotransporter SGLT1 expressed mainly in the intestine and kidney has been explored extensively for understanding the mechanism of sugar cotransport and its inhibition by a classical competitive inhibitor, phlorizin (Pz). It has been shown that inhibition of SGLT1 by Pz involves its interaction followed by major conformational changes in the Pz binding domain (PBD) in C-terminal loop 13. However, the mechanism of Pz inhibition and its interaction with other members of SGLT is not known. In this hypothesis, we performed molecular modeling of SGLT1-loop 13 with Pz and carried out primary sequence analyses and secondary structure predictions to determine qualitatively similar PBDs in C-termini of human SGLT2-4, except for vSGLT, which contains an unstructured short C-terminus. The ranking of predictions of Pz interaction strongly agrees with the following ranking of previously reported Pz inhibition: SGLT2>SGLT1>SGLT4>SGLT3>>vSGLT. In addition, the sugar binding residues were found to be quite conserved among all SGLT members investigated here. Based on these preliminary analyses, we propose that other Pz-sensitive SGLTs are also inhibited via mechanism similar to SGLT1 where an aglucone of Pz, phloretin, interacts with PBD and glucoside moiety with sugar binding residues. Our hypothesis sets the stage for future analyses on investigation of Pz interaction with SGLT family and further suggests that Pz modeling may be explored to design novel inhibitors targeting several SGLT members.

Pathogenic mutations causing glucose transport defects in GLUT1 transporter: The role of intermolecular forces in protein structure-function.

Two families of glucose transporter - the Na(+)-dependent glucose cotransporter-1 (SGLT family) and the facilitated diffusion glucose transporter family (GLUT family) - play a crucial role in the translocation of glucose across the epithelial cell membrane. How genetic mutations cause life-threatening diseases like GLUT1-deficiency syndrome (GLUT1-DS) is not well understood. In this review, we have combined previous functional data with our in silico analyses of the bacterial homologue of GLUT members, XylE (an outward-facing, partly occluded conformation) and previously proposed GLUT1 homology model (an inward-facing conformation). A variety of native and mutant side chain interactions were modeled to highlight the potential roles of mutations in destabilizing protein-protein interaction hence triggering structural and functional defects. This study sets the stage for future studies of the structural properties that mediate GLUT1 dysfunction and further suggests that both SGLT and GLUT families share conserved domains that stabilize the transporter structure/function via a similar mechanism.

Special interaction of anionic phosphatidic acid promotes high secondary structure in tetrameric potassium channel.

Anionic phosphatidic acid (PA) has been shown to stabilize and bind stronger than phosphatidylglycerol via electrostatic and hydrogen bond interaction with the positively charged residues of potassium channel KcsA. However, the effects of these lipids on KcsA folding or secondary structure are not clear. In this study, the secondary structure analyses of KcsA potassium channel was carried out using circular dichroism spectroscopy. It was found that PA interaction leads to increases in α-helical and ß-sheet content of KcsA protein. In PA, KcsA α-helical structure was further stabilized by classical membrane-active cosolvent trifluoroethanol followed by reduction in the ß-sheet content indicating cooperative transformation from the ß-sheet to an α-helical structure. The data further uncover the role of anionic PA in KcsA folding and provide mechanism by which strong hydrogen bonds/electrostatic interaction among PA headgroup and basic residues on lipid binding domains may induce high helical structure thereby altering the protein folding and increasing the stability of tetrameric assembly.

SLC5 and SLC2 transporters in epithelia-cellular role and molecular mechanisms.

Members of the SLC5 and SLC2 family are prominently involved in epithelial sugar transport. SGLT1 (sodium-glucose transporter) and SGLT2, as representatives of the former, mediate sodium-dependent uptake of sugars into intestinal and renal cells. GLUT2 (glucose transporter), as representative of the latter, facilitates the sodium-independent exit of sugars from cells. SGLT has played a major role in the formulation and experimental proof for the existence of sodium cotransport systems. Based on the sequence data and biochemical and biophysical analyses, the role of extramembranous loops in sugar and inhibitor binding can be delineated. Crystal structures and homology modeling of SGLT reveal that the sugar translocation involves operation of two hydrophobic gates and intermediate exofacial and endofacial occluded states of the carrier in an alternating access model. The same basic model is proposed for GLUT1. Studies on GLUT1 have pioneered the isolation of eukaryotic transporters by biochemical methods and the development of transport kinetics and transporter models. For GLUT1, results from extensive mutagenesis, cysteine substitution and accessibility studies can be incorporated into a homology model with a barrel-like structure in which accessibility to the extracellular and intracellular medium is altered by pinching movements of some of the helices. For SGLT1 and GLUT1, the extensive hydrophilic and hydrophobic interactions between sugars and binding sites of the various intramembrane helices occur and lead to different substrate specificities and inhibitor affinities of the two transporters. A complex network of regulatory steps adapts the transport activity to the needs of the body.

Structural insights into genetic variants of Na(+)/glucose cotransporter SGLT1 causing glucose-galactose malabsorption: vSGLT as a model structure.

Current advances in structural biology provide valuable insights into structure-function relationship of membrane transporters by solving crystal structures of bacterial homologs of human transporters. Therefore, scientists consider bacterial transporters as useful structural models for designing of drugs targeted in human diseases. The functional homology between Vibrio parahaemolyticus Na(+)/galactose transporter (vSGLT) and Na(+)/glucose cotransporter SGLT1 has been well established a decade ago. Now the crystal structure of vSGLT is considered quite valuable in explaining not only the cotransport mechanisms, but it also acts as a representative protein in understanding the protein stability and amino acid interactions within the core structure. We investigated the molecular mechanisms of genetic variations in SGLT1 that cause glucose-galactose malabsorption (GGM) defects using the crystal structure of vSGLT as a model sugar transporter. Our in silico mutagenesis and modeling analysis suggest that the GGM genetic variations lead to conformational changes either by structure destabilization or by formation of unnecessary interaction within the core structure of SGLT1 thereby explaining the genetic defects in Na(+) dependent sugar translocation across the cell membrane.

Transferring knowledge towards understanding the pore stabilizing variations in K(+) channels: pore stability in K(+) channels.

Recent advances in structural biology underlying mechanisms of channel gating have strengthened our knowledge about how K(+) channels can be inter-convertible between conductive and non-conductive states. We have reviewed and combined mutagenesis with biochemical, biophysical and structural information in order to understand the critical roles of the pore residues in stabilizing the pore structure and channel open state. We also discuss how the latest knowledge on the K(+) channel KcsA may provide a step towards better understanding of distinct pore stabilizing differences among diversified K(+) channels.

The ATP-sensitive K(+) channel ABCC8 S1369A type 2 diabetes risk variant increases MgATPase activity.

Pancreatic ß-cell ATP-sensitive K(+) (K(ATP)) channels are composed of Kir6.2 and SUR1 subunits encoded by the KCNJ11 and ABCC8 genes, respectively. Although rare monogenic activating mutations in these genes cause overt neonatal diabetes, the common variants E23K (KCNJ11) and S1369A (ABCC8) form a tightly heritable haplotype that is associated with an increased susceptibility to type 2 diabetes (T2D) risk. However, the molecular mechanism(s) underlying this risk remain to be elucidated. A homology model of the SUR1 nucleotide-binding domains (NBDs) indicates that residue 1369 is in close proximity to the major MgATPase site. Therefore, we investigated the intrinsic MgATPase activity of K(ATP) channels containing these variants. Electrophysiological and biochemical techniques were used to study the MgATPase activity of recombinant human K(ATP) channels or glutathione S-transferase and NBD2 fusion proteins containing the E23/S1369 (nonrisk) or K23/A1369 (risk) variant haplotypes. K(ATP) channels containing the K23/A1369 haplotype displayed a significantly increased stimulation by guanosine triphosphate compared with the E23/S1369 haplotype (3.2- vs. 1.8-fold). This effect was dependent on the presence of the A1369 variant and was lost in the absence of Mg(2+) ions or in the presence of the MgATPase inhibitor beryllium fluoride. Direct biochemical assays also confirmed an increase in MgATPase activity in NBD2 fusion proteins containing the A1369 variant. Our findings demonstrate that the A1369 variant increases K(ATP) channel MgATPase activity, providing a plausible molecular mechanism by which the K23/A1369 haplotype increases susceptibility to T2D in humans homozygous for these variants.

Diverse gating in K+ channels: differential role of the pore-helix glutamate in stabilizing the channel pore.

The selectivity filter and adjacent regions in the bacterial KcsA and inwardly rectifying K(+) (Kir) channels reveal significant conformational changes that cause the channel pore to transition from an activated to inactive state (C-type inactivation) once the channel is open. The meshwork of residues stabilizing the pore of KcsA involves Glu71-Asp80 carboxyl-carboxylate interaction 'behind' the selectivity filter. Interestingly, the Kir channels do not have this exact interaction, but instead have a Glu-Arg salt bridge where the Glu is in the same position but the Arg is one position N-terminal compared to the Asp in KcsA. Also, the Kir channels lack the Trp that hydrogen bonds to Asp80 in KcsA. Here, the sequence and structural information are combined to understand the dissimilarity in the role of the pore-helix Glu in stabilizing the pore structure in KcsA and Kir channels. This review illustrates that although Glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other; thereby suggesting a unique phenomenon of diverse gating patterns in K(+) channels.

Do small headgroups of phosphatidylethanolamine and phosphatidic acid lead to a similar folding pattern of the K(+) channel?

Phospholipid headgroups act as major determinants in proper folding of oligomeric membrane proteins. The K(+)-channel KcsA is the most popular model protein among these complexes. The presence of zwitterionic nonbilayer lipid phosphatidylethanolamine (PE) is crucial for efficient tetramerization and stabilization of KcsA in a lipid bilayer. In this study, the influence of PE on KcsA folding properties was analyzed by tryptophan fluorescence and acrylamide quenching experiments and compared with the effect of anionic phosphatidic acid (PA). The preliminary studies suggest that the small size and hydrogen bonding capability of the PE headgroup influences KcsA folding via a mechanism quite similar to that observed for anionic PA.

Improved technique for reconstituting incredibly high and soluble amounts of tetrameric K⁺ channel in natural membranes.

The reconstitution of large amounts of integral proteins into lipid vesicles is largely prompted by the complexity of most biological membranes and protein stability. We optimized a particular system which maximized the incorporation efficiency of large soluble amounts of KcsA potassium channel in Escherichia coli membranes. The effects of two detergents, octylglucoside and 3-[(cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), on KcsA reconstitution were compared. Reconstitution efficiency was found to be incredibly high for CHAPS-treated proteoliposomes followed by dialysis at room temperature. This approach may allow more accurate investigation of integral membrane proteins in their natural membrane environment via biophysical or biochemical techniques.

The potassium channel KcsA: a model protein in studying membrane protein oligomerization and stability of oligomeric assembly?

Many membrane proteins are functional as stable oligomers. An understanding of the conditions that elicit and enhance oligomerization is important in many therapeutics. In this regard, protein-protein and protein-lipid interactions play crucial roles in the assembly and stability of oligomeric complexes. Recent years have seen a rapid increase in the mechanistic information on the importance of cytoplasmic termini in determining subunit assembly and stability of oligomeric complexes. In addition, the role of specific protein-lipid interaction between anionic phospholipids and "hot spots" on the protein surface has also become evident in stabilizing oligomeric assemblies. This review focuses on several contemporary developments of membrane proteins that stabilize oligomers by taking the potassium channel KcsA as an exemplary ion channel.

A biophysical glance at the outer surface of the membrane transporter SGLT1.

Proteins mediating the transport of solutes across the cell membrane control the intracellular conditions in which life can occur. Because of the particular arrangement of spanning a lipid bilayer and the many conformations required for their function, transport proteins pose significant obstacles for the investigation of their structure-function relation. Crystallographic studies, if available, define the transmembrane segments in a "frozen" state and do not provide information on the dynamics of the extramembranous loops, which are similarly evolutionary conserved and thus as functionally important as the other parts of the protein. The current review presents biophysical methods that can shed light on the dynamics of transporters in the membrane. The techniques that are presented in some detail are single-molecule recognition atomic force microscopy and tryptophan scanning, which can report on the positioning of the loops and on conformational changes at the outer surface. Studies on a variety of symporters are discussed, which use gradients of sodium or protons as energy source to translocate (mainly organic) solutes against their concentration gradients into or out of the cells. Primarily, investigations of the sodium-glucose cotransporter SGLT1 are used as examples for this biophysical approach to understand transporter function.

The role of extramembranous cytoplasmic termini in assembly and stability of the tetrameric K(+)-channel KcsA.

Membrane-active alcohol 2,2,2-trifluoroethanol has been proven to be an attractive tool in the investigation of the intrinsic stability of integral membrane protein complexes by taking K(+)-channel KcsA as a suitable and representative ion channel. In the present study, the roles of both cytoplasmic N and C termini in channel assembly and stability of KcsA were determined. The N terminus (1-18 residues) slightly increased tetramer stability via electrostatic interactions in the presence of 30 mol.% acidic phosphatidylglycerol (PG) in phosphatidylcholine lipid bilayer. Furthermore, the N terminus was found to be potentially required for efficient channel (re)assembly. In contrast, truncation of the C terminus (125-160 residues) greatly facilitated channel reversibility from either a partially or a completely unfolded state, and this domain was substantially involved in stabilizing the tetramer in either the presence or absence of PG in lipid bilayer. These studies provide new insights into how extramembranous parts play their crucial roles in the assembly and stability of integral membrane protein complexes.

The role of phosphatidic acid and cardiolipin in stability of the tetrameric assembly of potassium channel KcsA.

In this study, the roles of two anionic phospholipids-phosphatidic acid (PA), which is an important signaling molecule, and cardiolipin (CL), which plays a crucial role in the bioenergetics of the cell-in stabilizing the oligomeric structure of potassium channel KcsA were determined. The stability of KcsA was drastically increased as a function of PA or CL content (mol%) in phosphatidylcholine (PC) bilayers. Deletion of the membrane-associated N terminus significantly reduced channel stability at high levels of PA content; however, the intrinsic stability of this protein was marginally affected in the presence of CL. These studies indicate that the electrostatic-hydrogen bond switch between PA and N terminus, involving basic residues, is much stronger than the stabilizing effect of CL. Furthermore, the unique properties of the PA headgroup alter protein assembly and folding properties differently from the CL headgroup, and both lipids stabilize the tetrameric assembly via their specific interaction on the extra- or the intracellular side of KcsA.

Monitoring the effects of strong cosolvent hexafluoroisopropanol in investigation of the tetrameric structure and stability of K(+)-channel KcsA.

Adsorption of small chain alcohols into lipid membranes significantly changes the conformational states of intrinsic membrane proteins. In this study, the effects of membrane-active strong cosolvent hexafluoroisopropanol (HFIP) on the intrinsic tetrameric stability of potassium channel KcsA were investigated. Presence of acidic phosphatidylglycerol (PG) in non-bilayer phosphatidylethanolamine (PE) or bilayer phosphatidylcholine (PC) significantly increased the tetrameric stability compared to zwitterionic pure PC bilayers. The stabilizing effect of PG in both lipid bilayers was completely abolished upon deletion of the membrane-anchored N-terminus. Tryptophan fluorescence and circular dichroism experiments indicated that HFIP destabilizes the tetramer possibly via drastic changes in the lateral pressure profile close to the membrane-water interface. The data suggest that HFIP disturbs the ionic, H-bonding and hydrophobic interactions among KcsA subunits where N-terminus presumably plays a crucial role in determining the channel proper folding and tetrameric structure via ionic/H-bond interactions between the helix dipole and the membrane lipids.

The "flipped" state in E71A-K+-channel KcsA exclusively alters the channel gating properties by tetraethylammonium and phosphatidylglycerol.

Mutation E71A in the bacterial K(+)-channel KcsA has been shown to abolish the activation-coupled inactivation of KcsA via significant alterations of the peptide backbone in the vicinity of the selectivity filter. In the present study, we examined channel-blocking behavior of KcsA-E71A by tetraethylammonium (TEA) from both the extra- and the intracellular sides. First, we found that E71A is inserted either in cis or trans orientation in a planar lipid bilayer; however, it exhibits only one orientation in proteoliposomes as determined by extravesicular partial chymotrypsin digestion. Second, E71A exhibits a lower extracellular TEA affinity and is more sensitive to intracellular TEA compared to wild-type KcsA, which apparently has >50-fold higher affinity for extracellular TEA and approximately 2.5-fold lower affinity for intracellular TEA compared to E71A. In additional experiments, we investigated the influence of negatively charged phosphatidylglycerol (PG) on channel-gating properties in phosphatidylcholine lipid bilayers. It was found that high PG content decreases the single-channel conductance and increases the channel open time and open probability. Taken together, our data suggest that the "flipped" conformation of the selectivity filter present in E71A allows weaker extracellular and stronger intracellular TEA binding, whereas higher PG content decreases channel conductivity and stabilizes the channel open "flipped" state via electrostatic interaction in the proximity of the channel pore.

Sodium permeability and sensitivity induced by mutations in the selectivity filter of the KcsA channel towards Kir channels.

The bacterial potassium (K(+)) channel KcsA provides an attractive model system to study ion permeation behavior in a selective K(+)-channel. We changed residue at the N-terminal end of the selectivity filter of KcsA (T74V) to its counterpart in inwardly rectifying K(+)-channels (Kir). The tetramer was found to be stable as unmodified KcsA. Under symmetrical and asymmetrical conditions, Na(+) increased the inward current in the virtual absence of K(+) however outward currents were nearly abolished which could be recovered upon internal K(+) addition. Na(+) also drastically increased the channel open time either in the presence or virtual absence of K(+). Furthermore, the T74V mutation decreased the internal Ba(2+) affinity of the channel possibly by binding to a K(+) site in the pore. In additional experiments, another point mutation V76I in T74V mutant was carried out thus the selectivity filter resembled more the selectivity filter of Kir channels. The mutant tetramer was converted into monomers as determined by conventional gel electrophoresis. However, native like gel electrophoresis, Trp fluorescence and acrylamide quenching experiments indicated that this mutant still formed a tetramer and apparently adopted similar folding properties as unmodified KcsA. Single-channel experiments further demonstrated that the channel was selective for K(+) over Na(+) as Na(+) blocked channel currents. These data suggest that single point mutation T74V alters the selectivity filter and allows simultaneous occupancy and conduction of K(+) and Na(+) probably via ion-ion interaction in the pore. In contrast, both mutations (T74V and V76I) in the same molecule seem to reorganize the pore conformation which controls the overall stability of a selective K(+)-channel.