Native State Research Articles

Se-Glargine: Chemical Synthesis of a Basal Insulin Analogue Stabilized by an Internal Diselenide Bridge.

Insulin has long provided a model for studies of protein folding and stability, enabling enhanced treatment of diabetes mellitus via analogue design. We describe the chemical synthesis of a basal insulin analogue stabilized by substitution of an internal cystine (A6-A11) by a diselenide bridge. The studies focused on insulin glargine (formulated as Lantus® and Toujeo®; Sanofi). Prepared at pH 4 in the presence of zinc ions, glargine exhibits a shifted isoelectric point due to a basic B chain extension (ArgB31 -ArgB32 ). Subcutaneous injection leads to pH-dependent precipitation of a long-lived depot. Pairwise substitution of CysA6 and CysA11 by selenocysteine was effected by solid-phase peptide synthesis; the modified A chain also contained substitution of AsnA21 by Gly, circumventing acid-catalyzed deamidation. Although chain combination of native glargine yielded negligible product, in accordance with previous synthetic studies, the pairwise selenocysteine substitution partially rescued this reaction: substantial product was obtained through repeated combination, yielding a stabilized insulin analogue. This strategy thus exploited both (a) the unique redox properties of selenocysteine in protein folding and (b) favorable packing of an internal diselenide bridge in the native state, once achieved. Such rational optimization of protein folding and stability may be generalizable to diverse disulfide-stabilized proteins of therapeutic interest.

An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

Biomechanical Comparison Between Double-Row Repair and Soft Tissue Tenodesis for Treatment of Proximal Rectus Femoris Avulsions.

Some patients with proximal rectus femoris (PRF) avulsions require surgical treatment after failed nonoperative treatment. There is no consensus on the superiority of suture anchor repair with the suture-bridge repair (SBR) technique versus tenodesis repair (TR) for PRF avulsions. To compare the failure load and elongation at failure between SBR and TR and to compare the stiffness of these 2 repair techniques versus the native state. Controlled laboratory study. Seven pairs of human cadaveric hemipelvises were dissected to the PRF and sartorius origins. Each specimen underwent preconditioning followed by a distraction test to determine the stiffness of the native specimen. One specimen of each pair received one of the repair methods (SBR or TR), while the other specimen in the pair received the other repair technique. After repair, each specimen underwent preconditioning followed by a pull to failure. The failure load, elongation at failure, stiffness, mode of failure, and stiffness as a percentage of the native state were determined for each repair. The SBR group exhibited a stronger failure load (223 ± 51 N vs 153 ± 32 N for the TR group; P = .0116) and significantly higher stiffness as a percentage from the native state (70.4% ± 19% vs 33.8% ± 15.5% for the TR group; P = .0085). While the stiffness of the repair state in the SBR group (41.5 ± 9.4 N/mm) was not significantly different from that of the native state (66.2 ± 36 N/mm), the stiffness of the repair state in the TR group (20.3 ± 7.5 N/mm) was significantly lower compared with that of the native state (65.4 ± 22.1 N/mm; P < .001) and repair state in the SBR group (41.5 ± 9.4 N/mm; P = .02). The SBR group primarily failed at the repair site (71%), and the TR group primarily failed at the suture-sartorius interface (43%) and at the muscle (29%). SBR and TR specimens were significantly weaker than the native tendon. The stiffness of the SBR was equivalent to that of the native tendon, while TR was significantly less stiff than the native tendon. The SBR was superior to TR in terms of failure load, stiffness, and percentage stiffness from the native state. SBR may be a better surgical option than TR to optimize failure load and stiffness for PRF avulsions.

Differential roles of C-3 and C-6 phosphate monoesters in affecting potato starch properties

The effects of starch phosphate monoester content (SPC), namely C-3 (C3P) and C-6 phosphate monoesters (C6P), on the starch properties were investigated using four potato starches with varied SPC/C3P/C6P and two non-phosphorylated maize starches with a similar range of amylose content (AC) as controls. The starch property results showed that a higher SPC is associated with lower turbidity, storage and loss modulus after storage, and water solubility, but higher swelling power (SP) and pasting viscosities. These findings suggested that SPC inhibited molecular rearrangement during storage and starch leaching during heating, and enhanced swelling and viscosities due to increased hydration and water uptake caused by the repulsion effect of phosphate groups and a less ordered crystalline structure. Increased SPC also resulted in lower resistant starch (RS) content in a native granular state but higher RS after retrogradation. Pearson correlations further indicated that SPC/C3P/C6P were positively correlated with peak (r2 = 0.925, 0.873 and 0.930, respectively), trough (r2 = 0.994, 0.968 and 0.988, respectively), and final viscosities (r2 = 0.981, 0.968 and 0.971, respectively). Notably, SPC, mainly C3P, exhibited a significantly positive correlation with SP (r2 = 0.859) and setback viscosity (r2 = 0.867), whereas SPC, mainly C6P, showed a weak positive correlation with RS after retrogradation (r2 = 0.746). However, SPC had no significant correlations with water solubility, turbidity and rheology properties, which were more correlated with AC. These findings are helpful for the food industry to select potato starches with desired properties based on their contents of SPC, C3P, or C6P.

From isolated polyelectrolytes to star-like assemblies: the role of sequence heterogeneity on the statistical structure of the intrinsically disordered neurofilament-low tail domain

Intrinsically disordered proteins (IDPs) are a subset of proteins that lack stable secondary structure. Given their polymeric nature, previous mean-field approximations have been used to describe the statistical structure of IDPs. However, the amino-acid sequence heterogeneity and complex intermolecular interaction network have significantly impeded the ability to get proper approximations. One such case is the intrinsically disordered tail domain of neurofilament low (NFLt), which comprises a 50 residue-long uncharged domain followed by a 96 residue-long negatively charged domain. Here, we measure two NFLt variants to identify the impact of the NFLt two main subdomains on its complex interactions and statistical structure. Using synchrotron small-angle x-ray scattering, we find that the uncharged domain of the NFLt induces attractive interactions that cause it to self-assemble into star-like polymer brushes. On the other hand, when the uncharged domain is truncated, the remaining charged N-terminal domains remain isolated in solution with typical polyelectrolyte characteristics. We further discuss how competing long- and short-ranged interactions within the polymer brushes dominate their ensemble structure and, in turn, their implications on previously observed phenomena in NFL native and diseased states.Graphic abstractVisual schematic of the SAXS measurement results of the Neurofilament-low tail domain IDP (NFLt). NFLts assemble into star-like brushes through their hydrophobic N-terminal domains (marked in blue). In increasing salinity, brush height (h) is initially increased following a decrease while gaining additional tails to their assembly. Isolating the charged sub-domain of the NFLt (marked in red) results in isolated polyelectrolytes

An expanded genetic code for native state, live labeling of prions

An expanded genetic code for native state, live labeling of prions

Anterior Latissimus Dorsi Transfer for Irreparable Subscapularis Tears Improves Shoulder Kinematics in a Dynamic Biomechanical Cadaveric Shoulder Model

Background: In young patients with irreparable subscapularis deficiency (SSC-D) and absence of severe osteoarthritis, anterior latissimus dorsi transfer (aLDT) has been proposed as a treatment option to restore the anteroposterior muscular force couple to regain sufficient shoulder function. However, evidence regarding the biomechanical effect of an aLDT on glenohumeral kinematics remains sparse. Purpose/Hypothesis: The purpose of this study was to investigate the effects of an aLDT on range of glenohumeral abduction motion, superior migration of the humeral head (SM), and cumulative deltoid force (cDF) in a simulated SSC-D model using a dynamic shoulder model. It was hypothesized that an aLDT would restore native shoulder kinematics by reestablishing the insufficient anteroposterior force couple. Study Design: Controlled laboratory study. Methods: Eight fresh-frozen cadaveric shoulders were tested using a validated shoulder simulator. Glenohumeral abduction angle (gAA), SM, and cDF were compared across 3 conditions: (1) native, (2) SSC-D, and (3) aLDT. gAA and SM were measured using 3-dimensional motion tracking, while cDF was recorded in real time during dynamic abduction motion by load cells connected to actuators. Results: The SSC-D significantly decreased gAA (Δ–9.8°; 95% CI, –14.1° to −5.5°; P < .001) and showed a significant increase in SM (Δ2.0 mm; 95% CI, 0.9 to 3.1 mm; P = .003), while cDF was similar (Δ7.8 N; 95% CI, –9.2 to 24.7 N; P = .586) when compared with the native state. Performing an aLDT resulted in a significantly increased gAA (Δ3.8°; 95% CI, 1.8° to 5.7°; P < .001), while cDF (Δ–36.1 N; 95% CI, –48.7 to −23.7 N; P < .001) was significantly reduced compared with the SSC-D. For the aLDT, no anterior subluxation was observed. However, the aLDT was not able to restore native gAA (Δ–6.1°; 95% CI, –8.9° to −3.2°; P < .001). Conclusion: In this cadaveric study, performing an aLDT for an irreparable subscapularis insufficiency restored the anteroposterior force couple and prevented superior and anterior humeral head migration, thus improving glenohumeral kinematics. Furthermore, compensatory deltoid forces were reduced by performing an aLDT. Clinical Relevance: Given the favorable effect of the aLDT on shoulder kinematics in this dynamic shoulder model, performing an aLDT may be considered as a treatment option in patients with irreparable SSC-D.

Do Not Waste Time─Ensure Success in Your Cross-Linking Mass Spectrometry Experiments before You Begin.

Cross-linking mass spectrometry (XL-MS) has become a very useful tool for studying protein complexes and interactions in living systems. It enables the investigation of many large and dynamic assemblies in their native state, providing an unbiased view of their protein interactions and restraints for integrative modeling. More researchers are turning toward trying XL-MS to probe their complexes of interest, especially in their native environments. However, due to the presence of other potentially higher abundant proteins, sufficient cross-links on a system of interest may not be reached to achieve satisfactory structural and interaction information. There are currently no rules for predicting whether XL-MS experiments are likely to work or not; in other words, if a protein complex of interest will lead to useful XL-MS data. Here, we show that a simple iBAQ (intensity-based absolute quantification) analysis performed from trypsin digest data can provide a good understanding of whether proteins of interest are abundant enough to achieve successful cross-linking data. Comparing our findings to large-scale data on diverse systems from several other groups, we show that proteins of interest should be at least in the top 20% abundance range to expect more than one cross-link found per protein. We foresee that this guideline is a good starting point for researchers who would like to use XL-MS to study their protein of interest and help ensure a successful cross-linking experiment from the beginning. Data are available via ProteomeXchange with identifier PXD045792.

Using Plantain Rachis Fibers and Mopa-Mopa Resin to Develop a Fully Biobased Composite Material.

A completely biobased composite material was developed using a matrix of natural resin extracted from the Elaegia pastoensis Mora plant, commonly known as Mopa-Mopa or "Barniz de Pasto", reinforced with fibers extracted from plantain rachis agricultural residues. A solvent process, involving grinding, distillation, filtration, and drying stages, was implemented to extract the resin from the plant bud. To obtain the resin from the plant bud, the vegetable material was ground and then dissolved in a water-alcohol blend, followed by distillation, filtration, and grinding until the powdered resin was ready for use in the preparation of the biocomposite. Likewise, using mechanical techniques, the plantain rachis fibers were extracted and worked in their native condition and with a previous alkalinization surface treatment. Finally, the biocomposite material was developed with and without incorporating stearic acid, which was included to reduce the material's moisture absorption. Ultimately stearic acid was used as an additive to reduce biocomposite moisture absorption. The tensile mechanical results showed that the Mopa-Mopa resin reached a maximum strength of 20 MPa, which decreased with the incorporation of the additive to 12 MPa, indicating its plasticization effect. Likewise, slight decreases in moisture absorption were also evidenced with the incorporation of stearic acid. With the inclusion of rachis plantain fibers in their native state, a reduction in the tensile mechanical properties was found, proportional to the amount added. On the other hand, with the alkalinization treatment of the fibers, the behavior was the opposite, evidencing increases in tensile strength, indicating that the fiber modification improved the interfacial adhesion with the Mopa-Mopa matrix. On the other hand, the evaluation of the moisture absorption of the biocomposite material evidenced, as expected, that the absorption level was favored by the relative humidity used in the conditioning (47, 77, and 97%), which also had an impact on the decrease of the mechanical tensile properties, being this was slightly counteracted by the inclusion of stearic acid in the formulation of the material.

4-Thiaproline accelerates the slow folding phase of proteins containing cis prolines in the native state by two orders of magnitude.

The cis/trans isomerization of peptidyl-prolyl peptide bonds is often the bottleneck of the refolding reaction for proteins containing cis proline residues in the native state. Proline (Pro) analogues, especially C4-substituted fluoroprolines, have been widely used in protein engineering to enhance the thermodynamic stability of peptides and proteins and to investigate folding kinetics. 4-thiaproline (Thp) has been shown to bias the ring pucker of Pro, to increase the cis population percentage of model peptides in comparison to Pro, and to diminish the activation energy barrier for the cis/trans isomerization reaction. Despite its intriguing properties, Thp has been seldom incorporated into proteins. Moreover, the impact of Thp on the folding kinetics of globular proteins has never been reported. In this study, we show that upon incorporation of Thp at cisPro76 into the thioredoxin variant Trx1P the half-life of the refolding reaction decreased from ~2 h to ~35 s. A dramatic acceleration of the refolding rate could be observed also for the protein pseudo wild-type barstar upon replacement of cisPro48 with Thp. Quantum chemical calculations suggested that the replacement of the Cγ H2 group by a sulfur atom in the pyrrolidine ring, might lower the barrier for cis/trans rotation due to a weakened peptide bond. The protein variants retained their thermodynamic stability upon incorporation of Thp, while the catalytic and enzymatic activities of the modified Trx1P remained unchanged. Our results show that the Pro isostere Thp might accelerate the rate of the slow refolding reaction for proteins containing cis proline residues in the native state, independent from the local structural environment.

The contribution of the ligamentum teres to the hip fluid seal: A biomechanics study

BackgroundThe suction seal of the hip plays an important role in maintaining hip stability; however, the function of the ligamentum teres in maintaining this seal remains poorly understood. This study aimed to evaluate the effectiveness of the hip suction seal in ligamentum teres deficient hips for joint positions occurring during gait. MethodsSix fresh-frozen human cadaveric hips were dissected and mounted to an Instron materials test system. Each specimen was analyzed for average peak distraction force, stiffness, and total energy during hip displacement. Testing was performed in the native intact ligamentum teres state and the deficient ligamentum teres state. Specimens were examined in 20° of flexion, neutral, and 10° of extension. FindingsIn the neutral position, the ligamentum teres deficient state displayed a significant decrease in peak distraction force (mean difference: 33.2 N, p < 0.001), average stiffness (mean difference: 63.7 N/mm, p = 0.016), and total energy (mean difference: 82.3 mJ, p = 0.022) compared to the intact controls. In extension, the deficient state exhibited a significant decrease in peak distraction force (mean difference: 42.8 N, p < 0.001) and total energy (mean difference: 72.9 mJ, p = 0.007). In flexion, the deficient state displayed a significant decrease in peak distraction force relative to contols (mean difference: 7.1 N, p = 0.003). InterpretationThe ligamentum teres plays a significant role in maintaining the suction seal of the hip, with its effect being most prominent when the hip is in neural alignment or in extension. The findings suggest that ligamentum teres deficiency may be a relevant treatment target in the clinical setting.

Insights into the pathogenesis of primary hyperoxaluria type I from the structural dynamics of alanine:glyoxylate aminotransferase variants.

Primary hyperoxaluria type I (PH1) is caused by deficient alanine:glyoxylate aminotransferase (AGT) activity. PH1-causing mutations in AGT lead to protein mistargeting and aggregation. Here, we use hydrogen-deuterium exchange (HDX) to characterize the wild-type (WT), the LM (a polymorphism frequent in PH1 patients) and the LM G170R (the most common mutation in PH1) variants of AGT. We provide the first experimental analysis of AGT structural dynamics, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants only show local destabilization. Enzymatic transamination of the pyridoxal 5-phosphate cofactor bound to AGT hardly affects stability. Our study, thus, supports that AGT misfolding is not caused by dramatic effects on structural dynamics.

Standardized analysis of syndesmosis stability in ankle trauma with an innovative syndesmosis-test-tool: a biomechanical study.

When treating ankle fractures, the question of syndesmosis complex involvement often arises. So far, there is no standardized method to reliably detect syndesmosis injuries in the surgical treatment of ankle fractures. For this reason, an intraoperative syndesmosis-test-tool (STT) was developed and compared to the recommended and established hook-test (HT). Tests were performed on cadaveric lower legs (n = 20) and the diastasis was visualized by 3D camera. Tests were performed at 50, 80, and 100N in native conditions and four instability levels. Instability was induced from anterior to posterior and the reverse on the opposite side. The impact on diastasis regarding the direction, the force level, the instability level, and the device used was checked using a general linear model for repeated measurement. The direction of the induced instability showed no influence on the diastasis during the stability tests. The diastasis measured with the STT increased from 0.5 to 3.0mm depending on the instability, while the range was lower with the HT (1.1 to 2.3mm). The results showed that the differentiation between the instability levels was statistically significantly better for the developed STT. The last level of maximum instability was significantly better differentiable with the STT compared to the HT. An average visualizable diastasis of more than 2mm could only be achieved at maximum instability. In conclusion, the newly developed STT was superior to the commonly used HT to detect instability.

KARR-seq reveals cellular higher-order RNA structures and RNA-RNA interactions.

RNA fate and function are affected by their structures and interactomes. However, how RNA and RNA-binding proteins (RBPs) assemble into higher-order structures and how RNA molecules may interact with each other to facilitate functions remain largely unknown. Here we present KARR-seq, which uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures inside cells, independent of local protein binding to RNA. KARR-seq depicts higher-order RNA structure and detects widespread intermolecular RNA-RNA interactions with high sensitivity and accuracy. Using KARR-seq, we show that translation represses mRNA compaction under native and stress conditions. We determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA-RNA interactions between the viruses and the host RNAs that potentially regulate viral replication.

Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

Host genotype, soil composition, and geo-climatic factors shape the fonio seed microbiome

BackgroundFonio (Digitaria exilis), an orphan millet crop, is the oldest indigenous crop in West Africa. Although the yield is low due to pre-domestication characteristics, the quick maturation time, drought tolerance, and the ability to thrive on poor soils make fonio a climate-smart crop. Being holobionts, plants evolve in close interaction with microbial partners, which is crucial for plant phenology and fitness. As seeds are the bottleneck of vertically transmitting plant microbiota, we proposed to unravel the seed microbiome of the under-domesticated and resilient crop fonio. Our study investigated the bacterial seed endophyte diversity across 126 sequenced fonio accessions from distinct locations in West Africa. We conducted a correlation study of the structures and functions of the seed-associated microbiomes with the native geo-climate and soil structure data. We also performed Genome-wide association studies (GWAS) to identify genetic loci associated with seed endophyte diversity.ResultWe report that fonio millet has diverse heritable seed endophytic taxa. We analyzed the seed microbiomes of 126 fonio accessions and showed that despite the diversity of microbiomes from distinct geographical locations, all fonio genetic groups share a core microbiome. In addition, we observed that native soil composition, geo-climatic factors, and host genotype correlate with the seed microbiomes. GWAS analysis of genetic loci associated with endophyte seed bacterial diversity identified fonio SNPs associated with genes functioning in embryo development and stress/defense response.ConclusionAnalysis of the seed endophyte of the climate-smart crop fonio indicated that despite possessing a heritable core microbiome, native conditions may shape the overall fonio seed microbiomes in different populations. These distinct microbiomes could play important roles in the adaptation of fonio to different environmental conditions. Our study identified the seed microbiome as a potential target for enhancing crop resilience to climate stress in a sustainable way.2rD2XEnkfTqrY_JSYv-2oiVideo

Media Discourse on the Kashmir Conflict after Abrogation of Article 370

The study tries to unfold how the Kashmir conflict is constructed in the Pakistani media after the abrogation of Article 370 of the Indian Constitution. To understand the media discourse a Critical discourse analysis of the news stories published about Indian held Kashmir (i.e. that part of the former princely state of Jammu and Kashmir occupied by India post the 1947 partition, notably the valley of Kashmir) in the e-paper of Dawn was carried out. The findings reveal a distinctive narrative strategy employed by Pakistani media in response to the abrogation of Article 370. This strategy accentuates the negative aspects of the Indian government's actions in Kashmir while simultaneously highlighting the voices of Kashmiris advocating for their right of self-determination. The findings also indicated that media discourse revolved around challenging the legitimacy of the Indian Parliament’s act of abrogating Article 370 and also focused on the casualties and losses which took place as a result of clashes between the Indian security forces and the Kashmiri separatist fighters. The media also gave significance to social media trends about Indian held Kashmir and also to posts of Kashmiri and Pakistani politicians. Negative actor description was used to highlight Bharatiya Janata Party (BJP)’s propaganda regarding the return of normalcy in the Indian held Kashmir whereas positive actor description was used for the Kashmiri separatist fighters.

G protein-coupled estrogen receptor (GPER)/GPR30 forms a complex with the β1-adrenergic receptor, a membrane-associated guanylate kinase (MAGUK) scaffold protein, and protein kinase A anchoring protein (AKAP) 5 in MCF7 breast cancer cells

G protein-coupled receptor 30 (GPR30), also named G protein-coupled estrogen receptor (GPER), and the β1-adrenergic receptor (β1AR) are G protein-coupled receptors (GPCR) that are implicated in breast cancer progression. Both receptors contain PSD-95/Discs-large/ZO-1 homology (PDZ) motifs in their C-terminal tails through which they interact in the plasma membrane with membrane-associated guanylate kinase (MAGUK) scaffold proteins, and in turn protein kinase A anchoring protein (AKAP) 5. GPR30 constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. We hypothesized that this inhibition is a consequence of a plasma membrane complex of these receptors. Using co-immunoprecipitation, confocal immunofluorescence microscopy, and bioluminescence resonance energy transfer (BRET), we show that GPR30 and β1AR reside in close proximity in a plasma membrane complex when transiently expressed in HEK293. Deleting the GPR30 C-terminal PDZ motif (-SSAV) does not interfere with the receptor complex, indicating that the complex is not PDZ-dependent. MCF7 breast cancer cells express GPR30, β1AR, MAGUKs, and AKAP5 in the plasma membrane, and co-immunoprecipitation revealed that these proteins exist in close proximity also under native conditions. Furthermore, expression of GPR30 in MCF7 cells constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. AKAP5 also inhibits β1AR-mediated cAMP production, which is not additive with GPR30-promoted inhibition. These results argue that GPR30 and β1AR form a PDZ-independent complex in MCF7 cells through which GPR30 constitutively and PDZ-dependently inhibits β1AR signaling via receptor interaction with MAGUKs and AKAP5.

Effect of Protein Surface Hydrophobicity and Surface Amines on Soy Adhesive Strength.

Soy is considered one of the most promising natural materials for manufacturing wood adhesives due to its low cost, high protein content, and ready availability. However, more cost-effective ways of improving its wet shear strength are needed to achieve wider market acceptance. Protein adhesive wet strength depends on the use of (typically expensive) crosslinking additives as well as the processing/denaturation of the protein. It has been commonly stated in the literature that protein denaturation leads to higher bond strength by activating the surface and exposing the reactive groups. Therefore, we investigated how differences in surface reactive groups (surface hydrophobicity and reactive amine groups) brought on with different denaturation treatments relate to bonding performance. Fourteen soy protein isolates (SPIs) with different denaturation histories were investigated. Characterization of the SPIs included surface hydrophobicity, surface amine content, extent of protein hydrolysis, and bond strength (wet and dry, with and without polyamidoamine epichlorohydrin (PAE) crosslinking agent) by ASTM D7998. The molecular weight patterns showed that proteins denatured by extensive hydrolysis had very low bond strengths. Adding the crosslinker, PAE, improved all the shear strength values. We found that the number of water-accessible reactive amine groups on protein surfaces had no impact on the adhesive strength, even with the amine-reactive crosslinker, PAE. Conversely, increased surface hydrophobicity was beneficial to adhesive strength in all cases, though this correlation was only statistically significant for wet strength without PAE. While, in general, denatured proteins are typically thought to form better bonds than native state proteins, this work suggests that it matters how proteins are denatured, and what surfaces become exposed. Denaturation by hydrolysis did not improve bond strength, and extensive hydrolysis seemed highly detrimental. Moreover, exposing hydrophobic surface groups was beneficial, but exposing covalent bond-forming reactive amine groups was not.

Unraveling the allosteric mechanisms of prolyl endopeptidases for celiac disease therapy: Insights from molecular dynamics simulations

Prolyl endopeptidases (PEP) from Sphingomonas capsulata (sc) and Myxococcus xanthus (mx) selectively degrade gluten peptides in vitro, offering a potential therapeutic strategy for celiac disease. However, the mechanisms governing the interaction of these enzymes with their substrates remain unclear. In this study, conventional molecular dynamics simulations with a microsecond timescale and targeted molecular dynamics simulations were performed to investigate the native states of mxPEP and scPEP enzymes, as well as their allosteric binding with a representative substrate, namely, Z-Ala-Pro-p-nitroanilide (pNA). The simulations reveal that the native scPEP is in an open state, while the native mxPEP is in a closed state. When pNA approaches a closed mxPEP, it binds to an allosteric pocket located at the first and second β-sheet of the β-propeller domain, inducing the opening of this enzyme. Neither enzyme is active in the open or partly-open states. Enzymatic activity is enabled only when the catalytic pocket in the closed state fully accommodates the substrates. The internal capacity of the catalytic pocket of PEP in the closed state determines the maximum size of the gluten peptides that the enzymes can catalyze. The present work provides essential molecular dynamics information for the redesign or engineering of PEP enzymes.