Pro Residues Research Articles

The Frequencies of Amino Acids in Secondary Structural Elements of Globular Proteins

Objective: The frequencies of amino acids in proteins for different structural levels have been determined by many studies. However, due to the different content of data sets, findings from these studies are inconsistent for some amino acids. This study aims to eliminate the contradictions in the findings of the studies by determining the frequencies of the amino acids in all structural level of globular proteins.
 Methods: The frequencies of the amino acids in overall protein, in secondary structural elements (helix, sheet, coil) and in subtypes of secondary structural elements (α-, π-, and 310-helices, and first, parallel and anti-parallel strands) were calculated separately using a data set including 4.882 dissimilar globular peptides. The frequencies of the amino acids were calculated as the ratio of the total number of a specific residue in related structure to the total number of all residues in the related structure.
 Results: The frequencies of residues determined in this study is partially in consistent with the other studies. The differences are probably due to the data set contents of the studies. The frequencies of the amino acids in subtypes of secondary structural elements were determined for the first time in this study. 
 Conclusions: Variations in the frequencies of PRO residue in 310-helix structure and of ILE, LEU, and VAL residues in strands of sheet structure are valuable findings for the improvement of secondary structure prediction methods, as they can be used as secondary structural elements markers.

On the interactions of peptides with gold nanoparticles: effects of sequence and size

Peptide-based self-assembly and synthesis techniques have emerged as a viable approach to designing active and stable inorganic nanostructures in aqueous media. In the present study, we use all-atom molecular dynamic (MD) simulations to study the interactions of ten short peptides (namely A3, AgBP1, AgBP2, AuBP1, AuBP2, GBP1, Midas2, Pd4, Z1, and Z2) with different gold nanoparticles (of different diameters ranging from 2 to 8 nm). Our MD simulation results imply that the gold nanoparticles have a remarkable effect on the stability and conformational properties of peptides. Moreover, the size of the gold nanoparticles and the type of peptide amino acid sequences play important roles in the stability of the peptide-AuNP complexes. Our results reveal that some amino acids such as Tyr, Phe, Met, Lys, Arg, and Gln have direct contact with the metal surface in comparison with Gly, Ala, Pro, Thr, and Val residues. The peptide adsorption on the surface of the gold nanoparticles is favorable from the energetic viewpoint, in which the van der Waals (vdW) interactions between the peptides and metal surface can be considered as one of the driving forces for the complexation process. The calculated Gibbs binding energies indicate that AuNPs have more sensitivity against the GBP1 peptide in the presence of different peptides. Overall, the results of this study can provide new insight into the peptide interaction with the gold nanoparticles from the molecular viewpoint, which can be important for designing new biomaterials based on the peptides and gold nanoparticles. Communicated by Ramaswamy H. Sarma

New insights into the bioaccessibility and metabolic fates of short-chain bioactive peptides in goat milk using the INFOGEST static digestion model and an improved data acquisition strategy

The metabolic fates of potentially bioactive short-chain peptides (SCPs; amino acid numbers between 2 and 4) in gastrointestinal digestion have received little attention due to their low concentration and broad suppression during high resolution mass spectrometry (HRMS) analysis. A tailored workflow integrating mesoporous magnetic solid phase extraction and a novel ion transmission strategy (data-dependent acquisition combined with both an inclusion list and an exclusion list followed by a data-independent acquisition) was used to profile the composition of SCPs during in vitro simulated digestion (LOQ 0.02 to 0.1 μg L-1). A total of 47 dipeptides, 59 tripeptides, and 21 tetrapeptides were identified and quantified from 0.01 to 27.84 mg L-1 (RSD ≤ 9.1%) based on parallel reaction monitoring and an internal standard method. The structural properties of stable SCPs resistant to intestinal digestion were determined by analysis of variance (p < 0.05), with a Pro residue at the C-terminal or penultimate position, a slightly greater negative charge at pH 7.0, and fewer C-terminal aliphatic and polar amino acids. SCPs' metabolic fates varied during digestion, but the overall trend of content change for either total or individual SCP increased as the digestion proceeded, and they were further assessed by a database-driven bioactivity search, which matched a wide variety of bioactivities with the predominance of dipeptidyl peptidase (DPP) IV and angiotensin-converting enzyme (ACE) inhibitors. This study facilitated the understanding of bioaccessibility of the food-derived SCPs and provided essential guidelines for the properties of conserved structure in vivo.

Carbene Cross-Linking in Gas-Phase Peptide Ion Scaffolds.

Scaffolds consisting of a peptide, a phthalate linker, and a 4,4-azipentyl group were synthesized and used to study intramolecular peptide-carbene cross-linking in gas-phase cations. Carbene intermediates were generated by UV-laser photodissociation at 355 nm of the diazirine ring in mass-selected ions, and the cross-linked products were detected and quantified by collision-induced dissociation tandem mass spectrometry (CID-MSn, n = 3-5). Peptide scaffolds containing Ala and Leu residues with a C-terminal Gly gave 21-26% yields of cross-linked products, while the presence of the Pro and His residues decreased the yields. Experiments using hydrogen-deuterium-hydrogen exchange, carboxyl group blocking, and analysis of CID-MSn spectra of reference synthetic products revealed that a significant fraction of cross-links involved the Gly amide and carboxyl groups. Interpretation of the cross-linking results was aided by Born-Oppenheimer molecular dynamics (BOMD) and density functional theory calculations that allowed us to establish the protonation sites and conformations of the precursor ions. Analysis of long (100 ps) BOMD trajectories was used to count close contacts between the incipient carbene and peptide atoms, and the counting statistics was correlated with the results of gas-phase cross-linking.

Structure-Activity Relationship Study of N-Hydroxyphtalimide Derivatives for the Detection of Amines during Fmoc-Solid-Phase Peptide Synthesis.

Previously, we developed a method for the detection of unprotected amino groups based on their reversible reaction with N-hydroxyphthalimide (NHPI) to form intensely colored products, which can be useful when conducting solid-phase peptide synthesis. Here, we describe a structure-activity relationship study of NHPI derivatives to identify the derivative best suited for this method using a spectrophotometer toward the estimation of chemical yields. We found that the products resulting from the reaction of the derivative with an unprotected amino group were only intensely colored if the structure of the derivative incorporated an NHPI framework. We also prepared five peptides, including those containing N-methyl and D-amino acid, and Pro residues, using our reversible detection method to detect unprotected amino groups. The mechanism of the detection reaction was also studied by the structural analysis of the NHPI (1) and diisopropylamine complex and concluded to entail salt formation between the N-hydroxy group and amine.

Membrane-Disrupting Activity of Cobra Cytotoxins Is Determined by Configuration of the N-Terminal Loop

In aqueous solutions, cobra cytotoxins (CTX), three-finger folded proteins, exhibit conformational equilibrium between conformers with either cis or trans peptide bonds in the N-terminal loop (loop-I). The equilibrium is shifted to the cis form in toxins with a pair of adjacent Pro residues in this loop. It is known that CTX with a single Pro residue in loop-I and a cis peptide bond do not interact with lipid membranes. Thus, if a cis peptide bond is present in loop-I, as in a Pro-Pro containing CTX, this should weaken its lipid interactions and likely cytotoxic activities. To test this, we have isolated seven CTX from Naja naja and N. haje cobra venoms. Antibacterial and cytotoxic activities of these CTX, as well as their capability to induce calcein leakage from phospholipid liposomes, were evaluated. We have found that CTX with a Pro-Pro peptide bond indeed exhibit attenuated membrane-perturbing activity in model membranes and lower cytotoxic/antibacterial activity compared to their counterparts with a single Pro residue in loop-I.

Evolutionarily Conserved Proline Residues Impede the Misfolding of the Mouse Prion Protein by Destabilizing an Aggregation-competent Partially Unfolded Form

The misfolding of the prion protein has been linked to several neurodegenerative diseases. Despite extensive studies, the mechanism of the misfolding process remains poorly understood. The present study structurally delineates the role of the conserved proline residues present in the structured C-terminal domain of the mouse prion protein (moPrP) in the misfolding process. It is shown that mutation of these Pro residues to Ala leads to destabilization of the native (N) state, and also to rapid misfolding. Using hydrogen–deuterium exchange (HDX) studies coupled with mass spectrometry (MS), it has been shown that the N state of moPrP is in rapid equilibrium with a partially unfolded form (PUF2*) at pH 4. It has been shown that the Pro to Ala mutations make PUF2* energetically more accessible from the N state by stabilizing it relative to the unfolded (U) state. The apparent rate constant of misfolding is found to be linearly proportional to the extent to which PUF2* is populated in equilibrium with the N state, strongly indicating that misfolding commences from PUF2*. It has also been shown that the Pro residues restrict the boundary of the structural core of the misfolded oligomers. Overall, this study highlights how the conserved proline residues control misfolding of the prion protein by modulating the stability of the partially unfolded form from which misfolding commences.

Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

Toxic Effect of Fullerene and Its Derivatives upon the Transmembrane β2-Adrenergic Receptors.

Numerous experiments have revealed that fullerene (C60) and its derivatives can bind to proteins and affect their biological functions. In this study, we explored the interaction between fullerine and the β2-adrenergic receptor (β2AR). The MD simulation results show that fullerene binds with the extracellular loop 2 (ECL2) and intracellular loop 2 (ICL2) of β2AR through hydrophobic interactions and π–π stacking interactions. In the C60_in1 trajectory, due to the π–π stacking interactions of fullerene molecules with PHE and PRO residues on ICL2, ICL2 completely flipped towards the fullerene direction and the fullerene moved slowly into the lipid membrane. When five fullerene molecules were placed on the extracellular side, they preferred to stack into a stable fullerene cluster (a deformed tetrahedral aggregate), and had almost no effect on the structure of β2AR. The hydroxyl groups of fullerene derivatives (C60(OH)X, X represents the number of hydroxyl groups, X = 4, 8) can form strong hydrogen bonds with the ECL2, helix6, and helix7 of β2AR. The hydroxyl groups firmly grasp the β2AR receptor like several claws, blocking the binding entry of ligands. The simulation results show that fullerene and fullerene derivatives may have a significant effect on the local structure of β2AR, especially the distortion of helix4, but bring about no great changes within the overall structure. It was found that C60 did not compete with ligands for binding sites, but blocked the ligands’ entry into the pocket channel. All the above observations suggest that fullerene and its derivatives exhibit certain cytotoxicity.

Post-Translationally Regulated Protein Arginine-to-Proline Conversion in Alzheimer's Brains.

The current belief is that amino acid sequences in protein structures are defined by DNA sequences. I challenge this concept by hypothesizing that an arginine (Arg) residue in the protein structure can post-translationally be converted to a proline (Pro) residue through a redox mechanism. Reactive oxygen species promote the formation of protein carbonylation, particularly on Arg and Pro residues, which both produce glutamyl semialdehyde. Our previous studies suggested that the Pro-to-glutamyl semialdehyde reaction could be reversible in the biological system, thereby opening up a pathway for the conversion of Arg to glutamyl semialdehyde by oxidation, and subsequently, to Pro by reduction in the protein structure. Our mass spectrometry and immunoblotting experiments provided evidence of the occurrence of the Arg-to-Pro conversion at position 108 (R108P) of the peroxiredoxin 6 (Prx6) protein in biological tissues and cells. In the human brain, Prx6 (R108P) occurs, and some Alzheimer’s brains exhibit increased Prx6 (R108P) levels, while others show decreased levels, indicating the complexity of redox processes in the disease state. I propose that Prx6 (R108P), as well as other post-translationally regulated protein Arg-to-Pro conversions, occur in the human body and play physiological and pathological roles.

High yield secretion of human erythropoietin from tobacco cells for ex vivo differentiation of hematopoietic stem cells towards red blood cells

Human erythropoietin (EPO) is a key cytokine in erythropoiesis by regulating differentiation of erythroid progenitor cells into red blood cells (RBCs). Plant cell cultures are considered as promising alternative bioproduction platforms for EPO. To overcome the bottlenecks of low protein productivity and secretion, EPO was expressed in tobacco BY-2 cells with a designer peptide tag, termed (SP)20 that consists of 20 tandem repeats of a “Ser-Pro” motif. This de novo designed tag directed extensive O-glycosylation on each Pro residue in plant cells and acted as a molecular carrier to promote the extracellular secretion of EPO. To facilitate the establishment of stable and high-expression BY-2 cell lines, EPO molecules were co-expressed with a reporter protein GFP, which could be used as a visual marker to monitor the protein expression during the subculture. The engineered (SP)20 glycomodule substantially increased the secreted yields of EPO up to 4.31 μg/mL. The (SP)20-tagged EPOs exhibited the expected activity in promoting the proliferation of TF-1 cells, though their EC50 was 12-fold higher than that of EPO standard. The (SP)20-tagged EPOs could also stimulate the ex vivo expansion and differentiation of hematopoietic stem cell (CD34+ cells) towards RBCs.

Molecular cloning, characterization, and homology modeling of serine hydroxymethyltransferase from psychrophilic bacterium Psychrobacter sp.

Serine hydroxymethyltransferase (SHMT) plays a significant role in the synthesis of l-serine, purine, and thymidylate, which could be extensively applied in the treatment of cancers and the development of antibiotics. In this study, cloned from Psychrobacter sp. ANT206, a novel cold-adapted SHMT gene (psshmt, 1257 bp) encoding a protein of 418 amino acids was expressed in Escherichia coli. The homology modeling result revealed that PsSHMT owned fewer Proline (Pro) residues and hydrogen bonds compared with its homologs from mesophilic E. coli and thermophilic Geobacillus stearothermophilus. In addition, the molecular weight of the purified recombinant PsSHMT (rPsSHMT) was identified to be 45 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, approximately. The enzymatic characteristics of the cold-adapted rPsSHMT displayed that its optimum temperature and pH were 30°C and 7.5, respectively, and its enzymatic activity could be inhibited by Cu2+ , significantly. rPsSHMT also showed a high kcat value and low ΔG at low temperatures. Furthermore, arginine (Arg) could affect the activity of rPsSHMT and be vital to its active sites. The results of this study reflected that these characteristics of the cold-adapted rPsSHMT made it a remarkable candidate that could be utilized in multiple industrial fields under low temperatures.

Nyuzenamide C, an Antiangiogenic Epoxy Cinnamic Acid-Containing Bicyclic Peptide from a Riverine Streptomyces sp.

A new nonribosomal peptide, nyuzenamide C (1), was discovered from riverine sediment-derived Streptomyces sp. DM14. Comprehensive analysis of the spectroscopic data of nyuzenamide C (1) revealed that 1 has a bicyclic backbone composed of six common amino acid residues (Asn, Leu, Pro, Gly, Val, and Thr) and four nonproteinogenic amino acid units, including hydroxyglycine, β-hydroxyphenylalanine, p-hydroxyphenylglycine, and 3,β-dihydroxytyrosine, along with 1,2-epoxypropyl cinnamic acid. The absolute configuration of 1 was proposed by J-based configuration analysis, the advanced Marfey's method, quantum mechanics-based DP4 calculations, and bioinformatic analysis of its nonribosomal peptide synthetase biosynthetic gene cluster. Nyuzenamide C (1) displayed antiangiogenic activity in human umbilical vein endothelial cells and induced quinone reductase in murine Hepa-1c1c7 cells.

The phytohormone abscisic acid modulates protein carbonylation in Arabidopsis thaliana.

Protein carbonylation is a post-translational modification associated with the reactive oxygen species. It results from the direct oxidation of the side chains of Lys, Arg, Pro, and Thr residues by hydroxyl radical HO• or the addition of reactive carbonyl species including α,β-unsaturated aldehydes and oxylipins to the side chain of Cys, His, and Lys. Recent findings indicated that the phytohormone abscisic acid (ABA) induces the production of α,β-unsaturated aldehydes that modulate the effect of ABA on stomatal closure. This indicated that α,β-unsaturated aldehydes might mediate ABA signaling. In this study, we investigated the ABA-induced protein carbonylation events by profiling the carbonylated proteome extracted from Arabidopsis thaliana leaves after ABA treatment. The carbonylated proteins were enriched by affinity chromatography and subjected to liquid chromatography-tandem mass spectrometry. We identified 180 carbonylated proteins. Of these, 26 proteins became carbonylated upon ABA treatment, whereas 163 proteins that were carbonylated in untreated samples were no longer detected in the ABA-treated samples, which points to dynamic control of protein carbonylation by ABA in A. thaliana. A few regulatory stress-related proteins and enzymes involved in the biosynthesis of the aspartate family of amino acids were overrepresented in the list of proteins, which the carbonylation status changed between untreated and ABA-treated samples. These results indicated that ABA triggers a change in the pattern of protein carbonylation in A. thaliana. This change is independent of the commonly seen increased levels of carbonylated proteins in the plants subjected to deadly stress conditions.

Relative configuration of Cys-Pro ester peptides in thioester formation.

A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C-terminus (CPE peptide) was transformed into a diketopiperazine (DKP) thioester via an intramolecular N-S acyl shift reaction and was then used for peptide ligation. The difference in reactivity between the CPE peptide stereoisomers was examined. In reactions of the CPE peptides that contained L-Cys-L-Pro or D-Cys-D-Pro, the desired DKP thioester was formed at the preceding amino acid residue. On the other hand, in reactions of the CPE peptides that contained D-Cys-L-Pro or L-Cys-D-Pro, a thiolactone was formed at the C-terminal prolyl ester, and the ligation occurred at the C-terminal Pro residue. Using this reaction, it was possible to efficiently prepare a cyclic peptide.

Purification and Characterization of a Novel Calcium-Binding Heptapeptide from the Hydrolysate of Tilapia Bone with Its Osteogenic Activity.

In this study, a calcium-binding peptide was obtained by hydrolyzing tilapia bone and its osteogenic activity was evaluated. Animal protease was selected from nine enzymes, and its hydrolysate was purified through preparative and semi-preparative reverse phase high-performance liquid chromatography. The purified peptide was identified as DGPSGPK (656.32 Da) and its calcium-binding capacity reached 111.98 µg/mg. The peptide calcium chelate (DGPSGPK-Ca) was obtained, and its structure was characterized through Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and mass spectrometry (MS). The results of XRD and SEM showed that DGPSGPK-Ca was formed as a new compound. The carboxyl and amino groups of Lys and Asp residues may be the chelating sites of DGPSGPK according to the FTIR and MS results. The molecular simulation showed the carbonyl groups of Asp, Pro, Ser, and Lys residues involved in the binding of calcium. The interaction of DGPSGPK and different integrins was evaluated by molecular docking simulation, and the main forces involved were electrostatic interaction forces, hydrogen bonding and hydrophobic interactions. Furthermore, DGPSGPK could inhibit the differentiation of osteoclast and promote the proliferation, differentiation and mineralization of osteoblasts.

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability.

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of "molecular surgery" to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical β-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual β-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level ("molecular surgery"), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of β-barrel fluorescent proteins.

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of "molecular surgery" to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical β-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual β-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level ("molecular surgery"), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of β-barrel fluorescent proteins.

Orai channel GOF mutants define critical functional distinctions between Orai subtypes

Store-operated calcium entry (SOCE) is a powerful mechanism controlling multiple physiological processes including transcription, cell proliferation, apoptosis, and secretion. PM Orai channels are activated by the ER Ca2+-sensing protein STIM, which induce channel conformational change that leads to gating. The gain of function (GOF) mutation Orai1-P245L, recognized to cause Stormorken-like syndrome, is known to have Ca2+ entry that’s independent of STIM activation due to the straightening of the Pro residue in the 4th transmembrane (TM4).

Antidiabetic Formulation Development Based on Natural Materials As α-Glucosidase Enzyme Inhibitor

Salam (Syzygium polyanthum), stevia (Stevia rebaudiana), tea (Camellia sinensis), and yakon (Smallanthus sonchifolius) leaves act as inhibitors of the α-glucosidase enzyme. The aims of this research were to (i) determine the best combination of polyherbals that could inhibit the α-glucosidase enzyme, (ii) determine the phytochemical content of the teraactive fraction, and (iii) predict potential bioactive compounds that were responsible for the inhibition of the α-glucosidase enzyme. The novelty of this research was its use of several types of herbs, combined with various compositions, to obtain the IC50 value. Also, this research predicted the activity of the compound through a molecular docking approach with Molegro Virtual Docker software. The extraction was carried out with the maceration method with 96% ethanol and then partitioned with ethyl acetate and water. Two fractions with high inhibition percentages were combined with a ratio of 1:1, 1:2, 2:1, 1:3, and 3:1, and they were tested against the α-glucosidase inhibitory enzyme. The best fraction was identified for chemical compound content by Liquid Chromatography Mass Spectroscopy (LC-MS/MS). The compounds were analyzed in silico by using Molegro Virtual Docker software, which obtained the best reranc score. The results showed that the ethanol extract of tea leaves had the highest activity, at 65.51%, and salam, stevia, and yakon leaves had inhibition percentages of 44.11%, 34.74%, and 28.85%, respectively. The combination of water fractions of tea and salam leaves had the best activity at a ratio of 3:1 with IC50 of 16.53 ppm, which was almost equivalent to acarbose as a positive control with IC50 of 14.62 ppm. The analysis of LC-MS/MS for the water fraction of tea contained nicotiflorin with m/z 594, a reranc score of -118.24, and amino acid residues Arg, Asp, Asn, Cys, Glu, His, Thr, and Tyr. The water fraction of salam leaves contained quercetagetin with m/z 319, the reranc score was –89.123, and the amino acid residues Asn, Asp, Gln, Lys, Phe, Pro, Thr, and Trp.