Charged Side Chains Research Articles

Versican controlled by Lmx1b regulates hyaluronate density and hydration for semicircular canal morphogenesis.

During inner ear semicircular canal morphogenesis in zebrafish, patterned canal-genesis zones express genes for extracellular matrix component synthesis. These include hyaluronan and the hyaluronan-binding chondroitin sulfate proteoglycan Versican, which are abundant in the matrices of many developing organs. Charged hyaluronate polymers play a key role in canal morphogenesis through osmotic swelling. However, the developmental factor(s) that pattern the synthesis of the matrix components and regulation of hyaluronate density and swelling are unknown. Here, we identify the transcription factor, Lmx1b, as a positive transcriptional regulator of hyaluronan, Versican, and chondroitin synthesis genes crucial for canal morphogenesis. We show that Versican regulates hyaluronan density through its protein core, whereas the charged chondroitin side chains contribute to the hydration of hyaluronate-ECM. Versican-tuned properties of hyaluronate matrices may be a broadly used mechanism in morphogenesis with important implications for understanding diseases where these matrices are impaired, and for hydrogel engineering for tissue regeneration.

Ordered assemblies of peptide nanoparticles with only positive charge

Surface charge patchiness of different charge types can influence the solution behaviours of colloidal particles and globular proteins. Herein, coiled-coil ‘bundlemer’ nanoparticles that display only a single type of surface charge (SC) are computationally designed to compare their solution behaviours to mixed charge-type (MC) counterparts with both positively and negatively charged side chains. Nematic and columnar liquid crystal phases are discovered in low concentrations of SC particles, indicative of particle end-to-end stacking into columns combined with lateral electrostatic repulsion between columns, while MC particles with the same net charge and particle shape produced only amorphous, soluble aggregates. Similarly, porous lattices are formed in mixtures of SC/MC particles of opposite charges while MC/MC mixtures of opposite charges produce only amorphous aggregates. The lattice structure is inferred with a machine learning optimization approach. The differences between SC and MC particle behaviours directly show the importance of surface electrostatic patchiness.

Likely Overstabilization of Charge-Charge Interactions in CHARMM36m(w): A Case for a99SB-disp Water.

Recent years have witnessed drastic improvements in general-purpose explicit solvent protein force fields, partially driven by the need to study intrinsically disordered proteins (IDPs), and yet the state-of-the-art force fields such as CHARMM36m (c36m) and a99SB-disp still provide different performances in simulating disordered protein states, where c36m has a bias toward overcompaction for large IDPs. Here, we examine the performance of c36m and a99SB-disp in describing the stabilities of a set of 46 amino acid backbone and side chain pairs in various configurations. The free energy results show that c36m systematically predicts stronger interactions compared to a99SB-disp by an average of 0.2 kcal/mol for nonpolar pairs, 0.6 kcal/mol for polar pairs, and 0.8 kcal/mol for salt bridges. The most severe overstabilization in c36m is observed for charged pairs involving the Arg and Glu side chains by up to 2.9 kcal/mol. Importantly, the systematic overstabilization of c36m is only marginally alleviated by c36mw, an ad hoc patch to c36m that increases the dispersion interactions between TIP3P hydrogens and protein atoms. Guided by free energy decomposition, we evaluated if revising the charges alone could alleviate the severe overstabilization of salt bridges of c36m(w) vs a99SB-disp. The results suggested that the direct modification of protein-water interactions is also necessary. Toward this end, we proposed a tentative modification to c36m, referred to as c36mrb-disp, which combines modified Arg side chain charges, retuned backbone hydrogen bonding strength, and the a99SB-disp water model. The modified force field successfully reproduces the secondary structures of several intrinsically disordered peptides and proteins including (AAQAA)3, GB1p, and p53 transactivation domain, while maintaining the stability of a set of folded proteins. This work provides a set of useful systems for benchmarking and optimizing protein force fields and highlights the importance of balancing protein-protein and protein-water electrostatic interactions for accurately describing both folded and disordered proteins.

A Rationally Designed Azobenzene Photoswitch for DNA G-Quadruplex Regulation in Live Cells.

G-quadruplex (G4) DNA structures are increasingly acknowledged as promising targets in cancer research, and the development of G4-specific stabilizing compounds may lay a fundamental foundation in precision medicine for cancer treatment. Here, we propose a light-responsive G4-binder for precise modulation of drug activation, providing dynamic and spatiotemporal control over G4-associated biological processes contributing to cancer cell death. We developed a specialized fluorinated azobenzene (AB) switch equipped with a quinoline unit and a positively charged carboxamide side chain, Q-Azo4F-C, designed for targeted binding to G4 structures within cells. Biophysical studies, combined with molecular dynamics simulations, provide insights into the unique coordination modes of the photoswitchable ligand in its trans and cis configurations when interacting with G4s. The observed variations in complexation processes between the two isomeric states in different cancer cell lines manifest in more than 25-fold reversible cytotoxic activity. Immunostaining conducted with the structure-specific G4 antibody (BG4), establishes a direct correlation between cytotoxicity and the varying extent of G4 induction regulated by the two isoforms. Finally, we demonstrate the photo-driven reversible regulation of G4 structures in lung cancer cells by Q-Azo4F-C. Our findings highlight the potential of light-responsive G4-binders in advancing precision cancer therapy through dynamic control of G4-mediated pathways.

A Rationally Designed Azobenzene Photoswitch for DNA G‐Quadruplex Regulation in Live Cells

AbstractG‐quadruplex (G4) DNA structures are increasingly acknowledged as promising targets in cancer research, and the development of G4‐specific stabilizing compounds may lay a fundamental foundation in precision medicine for cancer treatment. Here, we propose a light‐responsive G4‐binder for precise modulation of drug activation, providing dynamic and spatiotemporal control over G4‐associated biological processes contributing to cancer cell death. We developed a specialized fluorinated azobenzene (AB) switch equipped with a quinoline unit and a positively charged carboxamide side chain, Q‐Azo4F‐C, designed for targeted binding to G4 structures within cells. Biophysical studies, combined with molecular dynamics simulations, provide insights into the unique coordination modes of the photoswitchable ligand in its trans and cis configurations when interacting with G4s. The observed variations in complexation processes between the two isomeric states in different cancer cell lines manifest in more than 25‐fold reversible cytotoxic activity. Immunostaining conducted with the structure‐specific G4 antibody (BG4), establishes a direct correlation between cytotoxicity and the varying extent of G4 induction regulated by the two isoforms. Finally, we demonstrate the photo‐driven reversible regulation of G4 structures in lung cancer cells by Q‐Azo4F‐C. Our findings highlight the potential of light‐responsive G4‐binders in advancing precision cancer therapy through dynamic control of G4‐mediated pathways.

Poly(carboxylated ether)s as Cement Additives: The Effect of the Addition Method on Hydration Kinetics.

Polycarboxylate ether (PCE) superplasticisers have been widely used in cement formulations. However, it is not until recently that several studies have analysed the relationship between the properties and the molecular structure. In the present work, PCEs with different side chain lengths and charge densities synthesised through free radical copolymerisation are used to analyse the effect they have on the hydration of ordinary Portland cement (OPC). It was found that the addition method of these PCEs to the OPC significantly affects the hydration kinetics of the cement paste. When PCEs are added through the direct addition method, a linear dependency between the retardation of hydration and the microstructure of the used PCEs is observed. On the contrary, when PCEs are added through the delayed addition method (PCEs are added to the cement paste 5 min after water), no retardation in hydration is observed, but the rate of hydration is reduced.

Monitoring Binding of Protamine with DNA Using Protein Charge Transfer Spectra.

In this work, novel intrinsic electronic absorption (250-400 nm) with a molar extinction coefficient of 752 M-1cm-1 at 250 nm, arising from photoinduced electron transfer involving charged amino acid side chains and the polypeptide backbone, along with luminescence (300-500 nm) with quantum yield of 0.011 from subsequent charge recombination, was observed in salmon sperm Protamine (PRM). The absorption of PRM was attributed to the previously identified Peptide Backbone-to-Side chain Charge Transfer (PBS-CT) from the polypeptide backbone to the abundant cationic headgroups of Arginine in PRM, while the luminescence was believed to originate from charge recombination within the charge-separated excited states of PRM. Remarkably, since Arg is the only charged residue in the PRM sequence, the PRM Protein Charge Transfer Spectra (ProCharTS) is both totally and uniquely Arg specific. Interestingly, the peak of PRM luminescence emission spectrum was independent of the excitation wavelength, unlike other proteins such as human serum albumin, displaying unconventional luminescence. Aggregation-induced effects on PRM absorbance and luminescence were ruled out, as both PRM absorbance and luminescence increase maintained linearity with increasing concentration in the 25-150 μM range. Nucleoprotamine complex formation, resulting from the binding of PRM with calf-thymus genomic DNA (gDNA), was monitored through increased scattering by the nucleoprotamine complex, a decrease in gDNA/PRM absorbance, a decrease in gDNA/PRM ellipticity, and shifts of nucleoprotamine complex band upon agarose gel electrophoresis. Upon binding with gDNA (700 μM base pair concentration), PRM ProCharTS absorbance at 260 nm decreased by 72%. This decrease was attributed to the formation and subsequent precipitation of nucleoprotamine complex upon PRM-gDNA binding. The application of ProCharTS absorbance to indirectly monitor DNA-protein binding in a label-free approach was thus demonstrated.

A Few Charged Residues in Galectin-3's Folded and Disordered Regions Regulate Phase Separation.

Proteins with intrinsically disordered regions (IDRs) often undergo phase separation to control their functions spatiotemporally. Changing the pH alters the protonation levels of charged sidechains, which in turn affects the attractive or repulsive force for phase separation. In a cell, the rupture of membrane-bound compartments, such as lysosomes, creates an abrupt change in pH. However, how proteins' phase separation reacts to different pH environments remains largely unexplored. Here, using extensive mutagenesis, NMR spectroscopy, and biophysical techniques, it is shown that the assembly of galectin-3, a widely studied lysosomal damage marker, is driven by cation-π interactions between positively charged residues in its folded domain with aromatic residues in the IDR in addition to π-π interaction between IDRs. It is also found that the sole two negatively charged residues in its IDR sense pH changes for tuning the condensation tendency. Also, these two residues may prevent this prion-like IDR domain from forming rapid and extensive aggregates. These results demonstrate how cation-π, π-π, and electrostatic interactions can regulate protein condensation between disordered and structured domains and highlight the importance of sparse negatively charged residues in prion-like IDRs.

Qualitative and quantitative characterization of elastin-like polypeptides combining low-PH/low-temperature bottom-up and intact protein liquid chromatography mass spectrometric analysis.

Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer. Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications. Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs.

A Simple Binary Supramolecular Co-Assembly Platform for Enhanced Tumor Imaging and Therapy.

The primary challenges in tumor imaging and therapy revolve around improving targeting efficiency, enhancing probe/drug delivery efficacy, and minimizing off-target signals and toxicity. Although various carriers have been developed, many are difficult to synthesize, costly, and not universally applicable. Furthermore, numerous carriers exhibit limited delivery rates in solid tumors, particularly larger nanocarriers. To address these challenges, a simple binary co-assembly drug delivery platform has been designed using the readily synthesized small molecule Cys(SEt)-Lys-CBT (CKCBT) as the self-assembly building block. CKCBT can effectively penetrate tumor cells due to its positively charged Lys side chain and small size. Upon glutathione reduction, CKCBT co-assembles with Nile red or Chlorin e6 to form nanofibers inside tumor cells. This enables their specific accumulation in tumor cells rather than normal cells and extends their exposure time, resulting in precise and enhanced tumor imaging and treatment. Hence, this uncomplicated and highly efficient binary co-assembly drug delivery platform can be easily adapted to a broad spectrum of probes and drugs, presenting a novel approach for advancing clinical diagnosis and therapy.

Structural Reorganization Mechanism of the Aβ42 Fibril Mediated by N-Substituted Oligopyrrolamide ADH-353.

The inhibition of amyloid-β (Aβ) fibrillation and clearance of Aβ aggregates have emerged as a potential pharmacological strategy to alleviate Aβ aggregate-induced neurotoxicity in Alzheimer's disease (AD). Maity et al. shortlisted ADH-353 from a small library of positively charged N-substituted oligopyrrolamides for its notable ability to inhibit Aβ fibrillation, disintegrate intracellular cytotoxic Aβ oligomers, and alleviate Aβ-induced cytotoxicity in the SH-SY5Y and N2a cells. However, the molecular mechanism through which ADH-353 interacts with the Aβ42 fibrils, leading to their disruption and subsequent clearance, remains unclear. Thus, a detailed molecular mechanism underlying the disruption of neurotoxic Aβ42 fibrils (PDB ID 2NAO) by ADH-353 has been illuminated in this work using molecular dynamics simulations. Interestingly, conformational snapshots during simulation depicted the shortening and disappearance of β-strands and the emergence of a helix conformation, indicating a loss of the well-organized β-sheet-rich structure of the disease-relevant Aβ42 fibril on the incorporation of ADH-353. ADH-353 binds strongly to the Aβ42 fibril (ΔGbinding= -142.91 ± 1.61 kcal/mol) with a notable contribution from the electrostatic interactions between positively charged N-propylamine side chains of ADH-353 with the glutamic (Glu3, Glu11, and Glu22) and aspartic (Asp7 and Asp23) acid residues of the Aβ42 fibril. This aligns well with heteronuclear single quantum coherence NMR studies, which depict that the binding of ADH-353 with the Aβ peptide is driven by electrostatic and hydrophobic contacts. Furthermore, a noteworthy decrease in the binding affinity of Aβ42 fibril chains on the incorporation of ADH-353 indicates the weakening of interchain interactions leading to the disruption of the double-horseshoe conformation of the Aβ42 fibril. The illumination of key interactions responsible for the destabilization of the Aβ42 fibril by ADH-353 in this work will greatly aid in designing new chemical scaffolds with enhanced efficacy for the clearance of Aβ aggregates in AD.

First family with Perry syndrome from Mexico.

Perry syndrome (PS) is a rare autosomal dominant disease characterized by parkinsonism, central hypoventilation, weight loss and depression and is caused by pathogenic mutations in the dynactin subunit 1 (DCTN1) gene (encoding p150glued protein). To date, only two cases have been reported in Latin America, specifically in Colombia and Argentina. The present study, to the best of our knowledge, reports the first recorded Mexican family with PS. The clinical features of the proband and a family history of early parkinsonism led to the suspicion of PS. The pathogenic variant NM_004082:c.212G>A, causing a (p.Gly71Glu) mutation in the p150glued protein, was identified in exon 2 of the DCTN1 gene by exome sequencing, confirming the diagnosis of PS. (p.Gly71Glu) has been previously identified in at least 4 cases of PS from different ethnic backgrounds. Genetic counseling was provided to the available family members. To clarify the impact of the (p.Gly71Glu) variant on the structure and function of the cytoskeleton-associated protein Gly rich (CAP-Gly) domain of p150glued, Glu71 mutated CAP-Gly domains were modeled and compared with the wild-type. It was hypothesized that the larger and more charged side chain of Glu may induce conformational and electrostatic changes, imposing a conformational restriction on the peptide backbone that would affect interaction with the p150glued protein partners, causing dysfunction in the dynactin protein complex.

Structural Determinants of Peptide Nanopore Formation.

We have evolved the nanopore-forming macrolittin peptides from the bee venom peptide melittin using successive generations of synthetic molecular evolution. Despite their sequence similarity to the broadly membrane permeabilizing cytolytic melittin, the macrolittins have potent membrane selectivity. They form nanopores in synthetic bilayers made from 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) at extremely low peptide concentrations and yet have essentially no cytolytic activity against any cell membrane, even at high concentration. Here, we explore the structural determinants of macrolittin nanopore stability in POPC bilayers using atomistic molecular dynamics simulations and experiments on macrolittins and single-site variants. Simulations of macrolittin nanopores in POPC bilayers show that they are stabilized by an extensive, cooperative hydrogen bond network comprised of the many charged and polar side chains interacting with each other via bridges of water molecules and lipid headgroups. Lipid molecules with unusual conformations participate in the H-bond network and are an integral part of the nanopore structure. To explore the role of this H-bond network on membrane selectivity, we swapped three critical polar residues with the nonpolar residues found in melittin. All variants have potency, membrane selectivity, and cytotoxicity that were intermediate between a cytotoxic melittin variant called MelP5 and the macrolittins. Simulations showed that the variants had less organized H-bond networks of waters and lipids with unusual structures. The membrane-spanning, cooperative H-bond network is a critical determinant of macrolittin nanopore stability and membrane selectivity. The results described here will help guide the future design and optimization of peptide nanopore-based applications.

Enhancement of waterborne pathogen removal by functionalized biochar with ε-polylysine ″dynamic arms″: Potential application in ultrafiltration system

Widespread outbreaks of threatening infections caused by unknown pathogens and water transmission have spawned the development of adsorption methods for pathogen elimination. We proposed a biochar functionalization strategy involving ε-polylysine (PLL), a bio-macromolecular poly(amino acid)s with variable folding conformations, as a "pathogen gripper" on biochar. PLL was successfully bridged onto biochar via polydopamine (PDA) crosslinking. The extension of electropositive side chains within PLL enables the capture of both nanoscale viruses and micrometer-scale bacteria in water, achieving excellent removal performances. This functionalized biochar was tentatively incorporated into ultrafiltration (UF) system, to achieve effective and controllable adsorption and retention of pathogens, and to realize the transfer of pathogens from membrane surface/pore to biochar surface as well as flushing water. The biochar-amended UF systems presents complete retention (∼7 LRV) and hydraulic elution of pathogens into membrane flushing water. Improvements in removal of organics and anti-fouling capability were observed, indicating the broken trade-off in UF pathogen removal dependent on irreversible fouling. Chemical characterizations revealed adsorption mechanisms encompassing electrostatic/hydrophobic interactions, pore filling, electron transfer, chemical bonding and secondary structure transitions. Microscopic and mechanical analyses validated the mechanisms for rapid adsorption and pathogen lysis. Low-concentration alkaline solution for used biochar regeneration, facilitated the deprotonation and transformation of PLL side chain to folded structures (α-helix/β-sheet). Biochar regeneration process also promoted the effective detachment/inactivation of pathogens and protection of functional groups on biochar, corroborated by physicochemical inspection and molecular dynamics simulation. The foldability of poly(amino acid)s acting like dynamic arms, significantly contributed to pathogen capture/desorption/inactivation and biochar regeneration. This study also inspires future investigation for performances of UF systems amended by poly(amino acid)s-functionalized biochar under diverse pressure, temperature, reactive oxygen species of feeds and chemical cleaning solutions, with far-reaching implications for public health, environmental applications of biochar, and UF process improvement.

Insights into the function and regulation of the calcium-activated chloride channel TMEM16A

The TMEM16A channel, a member of the TMEM16 protein family comprising chloride (Cl−) channels and lipid scramblases, is activated by the free intracellular Ca2+ increments produced by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release after GqPCRs or Ca2+ entry through cationic channels. It is a ubiquitous transmembrane protein that participates in multiple physiological functions essential to mammals' lives. TMEM16A structure contains two identical 10-segment monomers joined at their transmembrane segment 10. Each monomer harbours one independent hourglass-shaped pore gated by Ca2+ ligation to an orthosteric site adjacent to the pore and controlled by two gates. The orthosteric site is created by assembling negatively charged glutamate side chains near the pore´s cytosolic end. When empty, this site generates an electrostatic barrier that controls channel rectification. In addition, an isoleucine-triad forms a hydrophobic gate at the boundary of the cytosolic vestibule and the inner side of the neck. When the cytosolic Ca2+ rises, one or two Ca2+ ions bind to the orthosteric site in a voltage (V)-dependent manner, thus neutralising the electrostatic barrier and triggering an allosteric gating mechanism propagating via transmembrane segment 6 to the hydrophobic gate. These coordinated events lead to pore opening, allowing the Cl− flux to ensure the physiological response. The Ca2+-dependent function of TMEM16A is highly regulated. Anions with higher permeability than Cl− facilitate V dependence by increasing the Ca2+ sensitivity, intracellular protons can replace Ca2+ and induce channel opening, and phosphatidylinositol 4,5-bisphosphate bound to four cytosolic sites likely maintains Ca2+ sensitivity. Additional regulation is afforded by cytosolic proteins, most likely by phosphorylation and protein-protein interaction mechanisms.

The methylome of motile cilia.

Cilia are highly complex motile, sensory, and secretory organelles that contain perhaps 1000 or more distinct protein components, many of which are subject to various posttranslational modifications such as phosphorylation, N-terminal acetylation, and proteolytic processing. Another common modification is the addition of one or more methyl groups to the side chains of arginine and lysine residues. These tunable additions delocalize the side-chain charge, decrease hydrogen bond capacity, and increase both bulk and hydrophobicity. Methylation is usually mediated by S-adenosylmethionine (SAM)-dependent methyltransferases and reversed by demethylases. Previous studies have identified several ciliary proteins that are subject to methylation including axonemal dynein heavy chains that are modified by a cytosolic methyltransferase. Here, we have performed an extensive proteomic analysis of multiple independently derived cilia samples to assess the potential for SAM metabolism and the extent of methylation in these organelles. We find that cilia contain all the enzymes needed for generation of the SAM methyl donor and recycling of the S-adenosylhomocysteine and tetrahydrofolate byproducts. In addition, we find that at least 155 distinct ciliary proteins are methylated, in some cases at multiple sites. These data provide a comprehensive resource for studying the consequences of methyl marks on ciliary biology.

Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties.

Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg.

Abstract 1021: Bulky Hydrophobic Side Chains In The β1-sandwich Of Microsomal Triglyceride Transfer Protein Are Critical For The Transfer Of Both Triglycerides And Phospholipids

Hyperlipidemia predispose individuals to cardio-metabolic diseases, the most common cause of global mortality. Microsomal triglyceride transfer protein (MTP) transfers multiple lipids and is essential for the assembly of apoB-containing lipoproteins. MTP inhibition lowers plasma lipids but causes lipid retention in the liver and intestine. Previous studies suggested two lipid transfer domains in MTP and that specific inhibition of triglycerides (TG) and not phospholipids (PL) transfer can lower plasma lipids without significant tissue lipid accumulation. However, how MTP transfers different lipids and the domains involved in these activities are unknown. Here, we tested a hypothesis that two different β-sandwich domains in MTP transfer TG and PL. Mutagenesis of charged amino acids in β2-sandwich had no effect on PL transfer activity indicating that they are not critical. In contrast, amino acids with bulky hydrophobic side chains in β1-sandwich were critical for both TG and PL transfer activities. Substitutions of these residues with smaller hydrophobic side chains or positive charges reduced, while negatively charged side chains severely attenuated MTP lipid transfer activities. These studies point to a common lipid transfer domain for TG and PL in MTP that is enriched with bulky hydrophobic amino acids. Furthermore, we observed a strong correlation in different MTP mutants with respect to loss of both the lipid transfer activities, again implicating a common binding site for TG and PL in MTP. We propose that targeting of areas other than the identified common lipid transfer domain might reduce plasma lipids without causing cellular lipid retention.

Intermediate molecular weight–fucosylated chondroitin sulfate from sea cucumber Cucumaria frondosa is a promising anticoagulant targeting intrinsic factor IXa

Thromboembolic diseases pose a serious risk to human health worldwide. Fucosylated chondroitin sulfate (FCS) is reported to have good anticoagulant activity with a low bleeding risk. Molecular weight plays a significant role in the anticoagulant activity of FCS, and FCS smaller than octasaccharide in size has no anticoagulant activity. Therefore, identifying the best candidate for developing novel anticoagulant FCS drugs is crucial. Herein, native FCS was isolated from sea cucumber Cucumaria frondosa (FCScf) and depolymerized into a series of lower molecular weights (FCScfs). A comprehensive assessment of the in vitro anticoagulant activity and in vivo bleeding risk of FCScfs with different molecule weights demonstrated that 10 kDa FCScf (FCScf-10 K) had a greater intrinsic anticoagulant activity than low molecular weight heparin (LMWH) without any bleeding risk. Using molecular modeling combined with experimental validation, we revealed that FCScf-10 K can specifically inhibit the formation of the Xase complex by binding the negatively charged sulfate group of FCScf-10 K to the positively charged side chain of arginine residues on the specific surface of factor IXa. Thus, these data demonstrate that the intermediate molecular weight FCScf-10 K is a promising candidate for the development of novel anticoagulant drugs.

Three novel umami peptides from watermelon soybean paste and the revelation of the umami mechanism through molecular docking with T1R1/T1R3

Watermelon soybean paste is a condiment popular in Henan, Shandong and Anhui provinces of China for the strong umami taste. This work used ultrafiltration and gel filtration chromatography to separate three novel umami peptides from watermelon soybean paste aqueous extract with sequences of AKEKFD, LAELK and LTFVER. The three peptides were all determined to be umami peptides by sensory evaluation and electronic tongue. Through molecular docking with umami receptor T1R1/T1R3, the umami taste mechanisms of the three umami peptides were revealed. The charged side chains in the umami peptides and T1R1/T1R3 played a key role in the perception of umami, and the primary factors of the interaction between umami peptides and T1R1/T1R3 were van der Waals forces and hydrogen bonds, which mainly contributed to the umami mechanism. This study will supplement the umami peptide database and offered a theoretical framework for further investigation on umami peptide.