ACS Omega

Wedelia chinensis is a medicinal herb of Asteraceae family. Preliminary phytochemical screening of internodal extract was done by using its aqueous, methanolic and ethanolic extracts. Preliminary phytochemical screening showed the presence of several pharmaceutically important compounds such as alkaloids, tannins, anthraquinons, saponins, flavonoids, terpenoids, phenols, etc. Aqueous internodal extract was used for the biosynthesis of gold nanoparticles. The characterization of gold nanoparticles were done by using a range of analytical techniques, such as energy-dispersive X-ray spectroscopy (EDS, EDX), X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transformed infrared spectroscopy (FTIR), scanning electron microscopy (SEM). The stability and other characteristics were ascertained using a UV-Vis spectrophotometer. The plasmon resonance occurred at the wavelength of 530 nm, thus showing the formation of gold nanoparticles. X-ray diffraction demonstrated the existence of Au-rich (fcc) phases in gold nanoparticles. FTIR analysis clearly indicated the participation of active constituents of internodal extract in the conversion of gold ions into AuNPs. DLS showed the size distribution (99.71 nm) of the suspended particles. There was variations in the sizes and shapes of the nanoparticles. The zeta potential of synthesized AuNPs was observed as -5.12 mV, thus showing the stability of the synthesized nanoparticles. In TEM study, uniform distribution of nanoparticles was seen with some agglomerates and average size of nanoparticles was observed in between 30-40 nm. The antioxidant potential of internodal extract was slightly higher than the synthesized gold nanoparticles. The green-synthesized AuNPs along with antibiotics demonstrated strong synergistic effect against several bacterial strains such as Escherichia coli and Bacillus subtilis. Gold nanoparticles showed good hemolytic activity against rat erythrocytes (90%). Gold nanoparticles also showed catalytic activity that reduces methylene blue at low concentration (1 mg). Highlights[tpltrtarr] Internodal extracts showed the presence of several active constituents in preliminary phytochemical screening. [tpltrtarr]Internodal extract was used for the synthesis of gold nanoparticles in one step process. [tpltrtarr]Synergistic action of AuNPs and antibiotics were reported against many pathogenic bacterial strains. [tpltrtarr]AuNPs showed its potential as antioxidant. [tpltrtarr]Hemolytic effect of nanoparticles was reported on rat blood sample. [tpltrtarr]Nanoparticles showed dye reducing activity thus enhancing its application in the purification of water bodies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/660090v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@12a27e3org.highwire.dtl.DTLVardef@a0d1b6org.highwire.dtl.DTLVardef@28178aorg.highwire.dtl.DTLVardef@216935_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig.C_FLOATNO Graphical abstract of AuNPs C_FIG

The spliceosome is a large ribonucleoprotein complex that regulates pre-mRNA splicing and has been an intriguing target for drug discovery. Essential to the assembly of the spliceosome are the small nuclear RNAs (snRNAs), which form RNA-RNA and RNA-protein interactions in the intact spliceosome and during its assembly. Here, we study the yeast U4/U6 snRNA assembly and report the rapid discovery of small molecule K-turn ligands via parallel small molecule microarray (SMM) screening of multiple related RNA constructs. For hit validation, biophysical analyses were conducted to confirm the binding and effects on thermodynamic stability and of RNA structure. One analog of the hit molecule (22) exhibited improved affinity towards the yeast U4/U6 snRNA (KD = 3.9 {+/-} 2.2 {micro}M). The specific interaction between 22 and the K-turn region was studied experimentally using deltaSHAPE and in silico with MD simulations. Moreover, this molecule was found to inhibit the binding of the U4 to Snu13 in biochemical assays (IC50 = 3.2 {+/-} 0.4 {micro}M). This work reports new ligands for the U4 snRNA and reveals that a multiplexed, structure-based approach can be used to identify small molecules that bind to specific regions of complex RNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/681657v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c156f4org.highwire.dtl.DTLVardef@133e90eorg.highwire.dtl.DTLVardef@109b793org.highwire.dtl.DTLVardef@f3301f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Altering the lectin properties by chemically synthesized glycoconjugates is important in glycobiology. A series of eight plant lectins with varying carbohydrate specificity were chosen as model systems to study the binding by synthetic glycoconjugates. One of our earlier paper 1 deals with the binding of glycoconjugates by jacalin. Further to this, we have now extended the studies to several other lectins having specificities towards glucose/mannose, galactose and lactose, and the results are reported in this paper on a comparative manner. The binding aspects were established by hemagglutination and fluorescence spectroscopy, and the conformational changes by CD spectroscopy. Out of the fourteen glycoconjugates used in the present study, a galactosyl-naphthyl derivative, 1c turns out to be most effective towards galactose-specific lectin in agglutination inhibition, fluorescence quenching by inducing considerable conformational changes. Similarly, mannosyl-naphthyl derivative, 3c turns out to be most effective in inhibiting the agglutination of Glc/Man specific lectins. Present study demonstrates differential recognition of conjugates towards lectins. The results also supported the existence of a correlation between the glycoconjugate and lectin specificity at the carbohydrate recognition domain (CRD). The glycoconjugate that inhibits the agglutination binds in the CRD via polar interactions as well as by nonpolar/hydrophobic interactions arising from the aromatic moiety of the conjugate, whereas, the non-inhibiting conjugates bind primarily via hydrophobic interactions. The specific and selective binding of the glycoconjugates by these lectins were proven by the docking studies. Thus, the present study has contributed immensely towards understanding the molecular interactions present between the lectins and small molecules that will eventually help better drug design where the presence of hydrophoibic moieties would play important role.

Different biochemical assays yield different rates of false positives than others either due to the nature of the enzyme, the technology associated with the assay, or properties of the compounds being screened. Ensuring that the right counter-screens are in place to identify false positives without wasting time and resources on them is of great importance. Herein we describe the results of a high throughput screen (HTS) against non-structural protein 3 (nsp3) protease PLPro, which resulted exclusively in false-positive hits. By triaging hit compounds through purification of metal chelating resin, we identified contamination by either copper or palladium as the most likely source of false positives from the library screening campaign. We then performed a systematic assessment of the vulnerability of nsp3 protease screening to metal contamination and evaluated common additives to combat the inhibitory effects of different metal salts. We further conducted a thorough survey of the literature reports of nsp3 HTS campaigns with a focus on the presence of additives and what metal susceptibility was likely, given the results of our work. We conclude that the majority of reported nsp3 screens are susceptible to copper contamination with a smaller proportion also potentially susceptible to palladium contamination.

Reducing sugar intake lowers the risk of obesity and associated metabolic disorders. Currently, this is achieved using artificial non-nutritive sweeteners, where their safety is widely debated and their contributions in various diseases is controversial. Emerging research suggests that these sweeteners may even increase the risk of cancer and cardiovascular problems, and some people experience gastrointestinal issues as a result of using them. A safer alternative to artificial sweeteners could be sweet-tasting proteins, such as brazzein, which do not appear to have any adverse health effects. In this study, protein language models were explored as a new method for protein design of brazzein. This innovative approach resulted in the identification of unexpected mutations, which opened up new possibilities for engineering thermostable and potentially sweeter versions of brazzein. To facilitate the characterization of the brazzein mutants, a simplified procedure was developed for expressing and analyzing related proteins. This process involved an efficient purification method using Lactococcus lactis (L. lactis), a generally recognized as safe (GRAS) bacterium, as well as taste receptor assays to evaluate sweetness. The study successfully demonstrated the potential of computational design in producing a more heat-resistant and potentially more palatable brazzein variant, V23.

Cocrystals have emerged as an exciting avenue in the pharmaceutical industry for altering the behavior of drugs while preserving the molecular structures of the active pharmaceutical ingredients (APIs). However, the preparation of pharmaceutical cocrystals remains relatively uncommon, presenting a potential opportunity for innovation. In this study, we developed cocrystals of Fleroxacin, a fluoroquinolone antibiotic belonging to the quinolone antibiotic class used in treating bacterial infections. Our approach involved co-crystallizing Fleroxacin with coformers (nicotinamide, salicylamide, and acetaminophen). The combination of Fleroxacin with different coformers allows us to explore chemical properties and the crystal efficacy. To achieve cocrystal formation, we employed a novel catalyst, pure glacial acetic acid, in conjunction with a ball milling machine. This methodology is particularly notable as it represents a first-time application of pure glacial acetic acid for co-crystallization and the co-crystallization of Fleroxacin. The cocrystals characteristics were analyzed using Synchrotron Powder X-ray Diffraction. The results showed the formation of novel cocrystals of Fleroxacin. The findings of this study contribute to the expanding body of knowledge on co-crystallization techniques and their potential applications in pharmaceutical development, especially for carboxylic acid-based drugs and drugs with very poor water solubilities but great permeabilities.

The inherently low thermal stability of RNA poses operational, accessibility, and financial challenges for RNA research and RNA-based technologies. Natural deep eutectic solvents (NADES) have been shown to enhance the stability of various macromolecules, including DNA and protein. This study explores NADES as alternative storage media for RNA preservation and evaluates their biocompatibility in human cells. In vitro-transcribed mRNA was stored in various NADES at temperatures ranging from 4-50 {degrees}C, and the integrity was assessed by quantifying its translatability, represented by protein-expressing cells. NADES effectively preserved RNA integrity, retaining over 50% of its translatability for at least four months at room temperature (21 {degrees}C) and 48 hours at 50 {degrees}C, in comparison to -80 {degrees}C storage. Notably, the preservation efficacy remained unaffected by temperature fluctuations. Furthermore, the selected NADES concentrations maintained [~] 99% cell viability, demonstrating their biocompatibility. These findings establish NADES as efficient, biocompatible RNA storage media, enabling stable storage and transport at ambient and extreme temperatures while withstanding sudden fluctuations. This enhanced stability simplifies and expands the accessibility of RNA-based applications. Additionally, the biocompatibility of NADES supports their potential use in RNA-based biomedical applications.

In this study, we describe the cytotoxic and antibacterial capabilities of gold nanoparticles (GNPs) synthesized from Dillenia indica leaves. Here, we used an environmentally friendly method to synthesize GNPs and then characterized them using techniques such as transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectroscopy, XRD, and Fourier transform infrared spectroscopy. The Surface Plasmon Resonance of GNPs was identified by an absorption peak at 533 nm in the UV-visible spectroscopic studies. The biosynthesized GNPs had an average size of 83 nm and were spherical in shape, according to TEM examination. Its interesting to note that the biosynthesized GNPs exhibited antibacterial action against a variety of bacterial isolates, both Gram-positive and negative. By using an MTT assay, growth inhibition and the cytotoxic effect of the synthesized GNPs against Daltons lymphoma cell lines were also evaluated.

Glaucoma is a known contributor to blindness in adults. This disease occurs as a result of overactivity of the carbonic anhydrase II (CAII) enzyme. Several of the commonly used sulfonamide drugs have been used to treat the symptoms of glaucoma but have been found to have low potency. To develop a more effective drug for the treatment of glaucoma, two novel CAII inhibitor drugs were designed through bioisoteric replacement and chemical intuition. This research discusses the development of these CAII inhibitor drugs from the existing glaucoma drug known as dichlorphenamide. The drugs created in this research were found to have a better docking score within the CAII binding site than that of dichlorphenamide. The drugs proposed in this paper would need to undergo further research to determine laboratory synthesis and potential clinical trialing.

Sm Endonuclease (SmEn) is a promiscuous, highly active nuclease widely used in protein purification, 2D protein gels, and gene and cell therapy. We aimed to recombinantly and economically produce this reagent using E. coli. Despite widespread application of E. coli for recombinant production of proteins, cytoplasmic expression of this protein resulted in no activity accumulation. We therefore investigated translocation of SmEn to the periplasm of E. coli by evaluating several signal sequences, E. coli host cells, and incubation conditions. For rapid feedback, we developed a crude lysate-based nuclease activity assay that enabled convenient screening and identified suitable conditions for active SmEn accumulation. Signal sequence selection was most influential with additional benefit gained by slowing synthesis either using the transcriptionally weakened strain, C43 (DE3) or by reducing incubation temperature. While our study provides valuable insights for optimizing a nuclease translocation and reducing production costs, more research is needed to explore the influence of mRNA secondary structure at the translation initiation region on protein expression and translocation. Overall, our rapid screening assay facilitated the development of an effective production process for a protein with potential cytoplasmic toxicity as well as the need of disulfide bond formation.

This article synthesized and characterized a novel hydrogel, which is formed using maleimide-thiol conjugation. Two precursors chitosan functionalized thiol groups and dextran functionalized maleimide groups prepared and characterized by NMR. The hydrogel studied by gelation time, swelling studies, viscoelastic properties, degradation rate. From gelation time result, we found that formed hydrogel gelation time could be changed with diffident weight percentage of precursors. Based on references, we found the best formular for the gelation and it was also determined for other studies. The swelling study indicated hydrogel has good flexibility and the degradation test indicated hydrogel is biodegradable. The viscoelastic test indicated hydrogel is elastic solid. From these studies, this a novel hydrogel could be potential for biomedical applications.

The dark genome comprising of non-expressing, non-translating, and extinct DNA sequences has remained a largely unexplored genomic space. Using computational and experimental approaches, novel insights into the dark matter genome have recently been gained, revealing the presence of a vast and unexplored resource. Non-coding RNA (ncRNA) refers to a class of RNA molecules that do not encode proteins but play important regulatory roles in the cell. We asked if it was possible to make functional peptides and proteins from ncRNA leading to a new biological insight and applications? Here we present initial computational data in support of making functional noncoding proteins (NCP) from ncRNA sequences. Different types of non-coding genomic sequences originating from Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens were studied to understand sequence composition, secondary structure, and physiochemical properties of NCPs. This work builds the foundation for experimentally characterizing the first-in-the-class non-coding proteins leading to a novel insights and applications.

The growing resistance of microbes to conventional antibiotics and the environmental impact of chemical synthesis methods highlight the urgent need for effective and sustainable antimicrobial agents. In this study, we address these challenges by synthesizing silver nanoparticles (AgNPs) using Ocimum sanctum leaf extract, a green and efficient method. We confirmed AgNPs synthesis through UV-vis spectroscopy, observing a Surface Plasmon Resonance (SPR) peak centered at 420 nm. X-ray Diffraction (XRD) analysis showed intense peaks at 37.81{degrees}, 45.01{degrees}, 63.91{degrees}, and 78.0{degrees}, corresponding to Braggs reflections at 111, 200, 220, and 311, respectively, with an average particle size of approximately 18 nm calculated using Scherrers formula. The reduction of Ag+ was monitored using Atomic Absorption Spectroscopy (AAS), which revealed a rapid decrease from 2.73 ppm to 0.023 ppm within four minutes, driven by the active reducing agents in the O. sanctum extract. The Scanning Electron Microscope (SEM) image confirmed the spherical shape of the AgNPs, with an average size of 23.82 {+/-} 4.17 nm. Fourier Transform Infrared (FTIR) spectroscopy identified absorption peaks at 1635 cm-1 and 3430 cm-1, associated with the amide I bond of proteins and OH stretching in alcohols and phenolic compounds, respectively. The synthesized AgNPs exhibited significant antimicrobial activity, demonstrated by a dose-dependent inhibitory effect against bacterial strains. The inhibition zone measured 6 mm at the lowest concentration of 5 {micro}g/L and increased progressively to 14 mm at the highest concentration of 25 {micro}g/L, indicating strong antimicrobial potential even at low dosages.

AO_SCPLOWBSTRACTC_SCPLOWWe have developed Fullerene-C60 nanoformulations containing discrete sized nanoparticles by dispersing concentration range of Fullerene. Small sized particles are cytotoxic while larger ones are cell proliferative. The cell proliferative property is used for tissue repair in cellular and animal wound models.

Carbohydrate hydrolysing enzymes assume special industrial and commercial interest as a source for yielding fermentable glucose especially for the biofuel industry. Among these enzymes, the exo-{beta}-(1,3) glucanases are promising for industrial use as they hydrolyze sugars such as laminarin, a major constituent of the algal cell wall. Exploring the structure and function of these enzymes is of particular interest for the improvement of their functional properties for industrial use. We report the structural and biochemical characterizations of Aspergillus oryzae exo-{beta}-(1,3) glucanase (AoBgl). We have expressed, purified, and performed biochemical characterizations of the recombinant AoBgl. Purified AoBgl is found to hydrolyse {beta}-(1,3)-glycosidic linkages present in the oligosaccharide laminaritriose and the polysaccharide, laminarin effectively while retaining >50% activity at glucose concentrations of around 1.5M. We have determined three high-resolution structures of AoBgl: (a) apo form at 1.75 [A], (b) complexed form with bound disaccharide at 1.73 [A] and (c) glucose-bound form at 1.20 [A]. Sequence analysis and structural comparison indicate that AoBgl belongs to the GH5 sugar hydrolase family. The sugar-bound structures reveal the mode of substrate binding and interactions at the active site of AoBgl. Further, molecular dynamics simulation and mutational studies indicate that AoBgl can effectively bind trisaccharides and higher oligosaccharides. Our biochemical and structural data provide detailed molecular insights into the active site of this GH5 enzyme and would be helpful in the rational engineering of glycosyl hydrolases belonging to similar families for industrial use.

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins, making it an interesting for many biotechnology applications. The discovery of bacterial glycosylation mechanisms, opened a new perspective and transfer of C.jejuni N-linked glycosylation into laboratory work-horse E. coli increased research pace in the field exponentially. It has been previously showed that utilizing N-Linked Glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. In this study, we utilized N-linked Glycosylation to glycosylate alkaline phosphatase (ALP) enzyme in E. coli and investigate the effects of glycosylation on an enzyme. Considering the glycosylation mechanism is highly dependent on the acceptor protein, ALP constructs carrying glycosylation tag at different locations of the gene has been created and glycosylation rates have been calculated. The most glycosylated construct has been selected for comparison with the native enzyme. We investigated the performance of glycosylated ALP in terms of its thermostability, proteolytic stability, tolerance to suboptimal pH and under denaturing conditions. Studies showed that glycosylated ALP performed remarkably better at optimal and harsh conditions Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.

This study dealt with nanonization of eugenol, a major phytochemical present in basil leaf, which has pharmacological potential as an anti-bacterial agent. Eugenol nanoparticle (ENP) was synthesized by simple ultrasonic cavitation method through emulsification of hydrophobic eugenol into hydrophilic gelatin. Thus, the nanonization process made the water-insoluble eugenol to water-soluble nano-eugenol, making the nano-form bioavailable. The average size of the ENPs was 20-30 nm. Entrapment efficiency of eugenol within gelatin cap was about 80% of the eugenol, that was used as precursor in the nanonization reaction. In vitro release of eugenol from gelatin cap was slow and sustained over a period of five days. The ENP had higher anti-biofilm potency than eugenol for both formation and eradication of biofilm, formed by clinically relevant pathogen Pseudomonas aeruginosa. Minimal biofilm inhibitory concentration and minimal biofilm eradication concentration of ENPs were 2.0 and 4.0 mM respectively. In addition, the measurement of P. aeruginosa biofilm biomass, biofilm pellicle formation, biofilm thickness, amount of biofilm-forming extra-polymeric substance, cell surface hydrophobicity, cell swarming and twitching efficiencies, cellular morphology and biofilm formation in catheter demonstrated that the anti-biofilm efficacy of nano-eugenol was 30-40% higher than that of bulk eugenol. Thus, ENP can be used as a potential drug against pneumonia, a chronic infection in lung caused by P. aeruginosa, which is difficult to treat with antibiotics, due to natural intrinsic resistance of biofilm-formed cells to most antibiotics. The overall actions of ENP have been presented in the figure 1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/521144v1_fig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@12445e2org.highwire.dtl.DTLVardef@6813aforg.highwire.dtl.DTLVardef@e4779borg.highwire.dtl.DTLVardef@1688a9a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Graphical abstract showing overall anti-biofilm action of ENP that is capable to inhibit each and every step of biofilm formation. ENP also carries the potential to destroy EPS and kill the biofilm residing cells by membrane disruption. Bacterial colonization has been found to be prevented significantly on ENP-coated catheter. C_FIG HighlightsO_LINano-formulation of eugenol, an important phytochemical, by ultrasonic cavitation method, which was simple, time-saving, low-cost and eco-friendly. C_LIO_LINanonization made water-insoluble eugenol into water-soluble form with enhanced therapeutic efficacy. C_LIO_LIThe eugenol nanoparticle (ENP) could inhibit formation of biofilm as well as facilitate eradication of pre-formed biofilm of P. aeruginosa. C_LIO_LIBiofilm formation was found to be prevented significantly on ENP-coated catheter. C_LIO_LINano-eugenol may be used as a potential drug against bacterial diseases, caused by pseudomonal biofilm, which are difficult to treat by antibiotics. C_LIO_LINano-formulated eugenol may also be used as an effective anti-fouling agent for biomedical devices like contact lens, pace-maker, materials for organ transplantation etc. to prevent bacterial colonization. C_LI

Specific phosphorylation patterns control the activity of multiphosphorylated proteins. In case of the Tau protein, multiphosphorylation leads to the formation of different disease-related condensates and aggregates. Studying the role of these specific patterns at the protein level is crucial for understanding the molecular mechanisms of Tauopathies such as Alzheimers Disease. However, due to the extreme difficulty in obtaining recombinant proteins with specific phosphorylation patterns using kinase-based methods, it is practically impossible to study the connection between specific phosphorylation patterns and aggregation events at the protein level. Here we addressed this problem by reducing the system to the peptide level and studying the effect of specific phosphorylation patterns on the condensation and aggregation of a specific domain of Tau, R4 (residues 336-358). To achieve this aim, we have applied advanced methods to synthesize a library of multiphosphorylated peptides derived from R4. We showed that specific phosphorylation patterns stringently control the formation of Tau aggregates and condensates. Phosphorylation of Ser341 promoted aggregation of R4 while phosphorylation of Ser352 promoted its condensation. Interestingly, Ser356 phosphorylation inhibited both processes, which can be overridden by double-phosphorylation at Ser341/Ser352. Differences between the microenvironments of the phosphorylated residues lead to their different effects on R4 aggregation upon phosphorylation. Our results show that working at the domain level using advanced peptide synthesis methods is a highly useful and practical way to provide valuable information about the effects of post translational modifications on protein activity.

Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that "Candidatus Accumulibacter" can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a "Ca. Accumulibacter" enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of "Ca. Accumulibacter" contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with "Ca. Accumulibcater" and used size-exclusion chromatography to separate them into different molecular weight fractions. This separation resulted in two high molecular weight (> 5,500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This suggest that they can serve as source for sepsis treatment drugs, although further purification needs to be evaluated. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/508216v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1f59ff7org.highwire.dtl.DTLVardef@d933fcorg.highwire.dtl.DTLVardef@1e3ff78org.highwire.dtl.DTLVardef@1994948_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. C_LIO_LISize exclusion chromatography allows to obtain high molecular weight polysaccharide-rich fractions enriched with NulOs. C_LIO_LIEPS and the NulOs-rich fractions can serve as source for sepsis treatment drugs. C_LI

{gamma}-Secretase is an intramembrane protease complex, with nearly 150 substrates that are cleaved within their transmembrane domains (TMD). Amyloid Precursor Protein (APP) is the most widely studied, as processive proteolysis by {gamma}-secretase releases the amyloid-{beta}-peptide (A{beta}) implicated in the pathogenesis of Alzheimers disease. In contrast, proteolysis of other substrates has been little explored. The only known sequence specificity rule for {gamma}-secretase cleavage is for APP, in which phenylalanine is not tolerated at P2 with respect to any step in processive proteolysis. Recently, we found this specificity rule applies to the initial cleavage of Notch1 substrate as well. In this study, we examined the site of initial cleavage by {gamma}-secretase and explored the phenylalanine rule for two other {gamma}-secretase substrates: neuregulin1 (NRG1) and E-cadherin (CDH1). Upon incubation of recombinant substrates with purified protease complex, followed by mass spectroscopy (MS) and immunoblot analysis, initial cleavage products for NRG1 and CDH1 were identified. Two cleavage sites were observed in the NRG1 TMD, one of which matched that seen previously. However, the observed single CDH1 TMD cleavage site differed from the reported cytosolic cleavage site. Phenylalanine mutants of NRG1 and CDH1 in the P2 position relative to the first {gamma}-secretase cleavage site showed a shift in the cleavage site, along with reduction in total C-terminal and N-terminal products, compared to that seen with wild-type substrates. Taking together, these findings clarify the initial cleavage sites of NRG1 and CDH1 and support the intolerance of Phe at P2 position as a general rule for {gamma}-secretase substrates. For Table of Contents use only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/649652v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1cfdda7org.highwire.dtl.DTLVardef@1e10813org.highwire.dtl.DTLVardef@d94ba6org.highwire.dtl.DTLVardef@1f58c08_HPS_FORMAT_FIGEXP M_FIG C_FIG