Parent Protein Research Articles

Invited review: Dairy proteins and bioactive peptides: Modeling digestion and the intestinal barrier

Dairy products are one of the most important sources of biologically active proteins and peptides. The health-promoting functions of these peptides are related to their primary structure, which depends on the parent protein composition. A crucial issue in this field is the demonstration of a cause-effect relationship from the ingested protein form to the bioactive form in vivo. Intervention studies represent the gold standard in nutritional research; however, attention has increasingly been focused on the development of sophisticated in vitro models of digestion to elucidate the mechanism of action of dairy nutrients in a mechanistic way and significantly reduce the number of in vivo trials. On the other hand, the epithelial intestinal barrier is the first gate that actively interacts with digestion metabolites, making the intestinal cells the first target tissue of dairy nutrients and respective metabolites. An evolution of the in vitro digestion approach in the study of dairy proteins and derived bioactive compounds is the setup of combined in vitro digestion and cell culture models taking into consideration the endpoint to measure the target organism (e.g., animal, human) and the key concepts of bioaccessibility, bioavailability, and bioactivity. This review discusses the relevance and challenges of modeling digestion and the intestinal barrier, focusing on the implications for the modeling of dairy protein digestion for bioactivity evaluation.

108: Molecular similarities and dissimilarities between hellp syndrome and preeclampsia (PE) using shotgun and targeted proteomics

Although clinical management of HELLP syndrome is similar to PE, hypertension and proteinuria do not always occur with HELPP. Thus, it was suggested that HELLP and PE could be separate clinical entities. Based on the ability of the urine misfolded proteins to retain Congo red (congophilia) it was suggested PE is a protein conformational disorder similar to Alzheimer’s disease. Molecular diagnostics are becoming more often part of modern clinical medicine. Here we used state of the art shotgun proteomics and targeted multiple reaction monitoring (MRM) mass spectrometry to discover and validate molecular signatures in crude urine samples of pregnant women with PE and HELLP that potentially differentiate between these two clinical conditions. We analyzed urine samples from six groups of patients classified phenotypically as: non-hypertensive healthy controls (CRL, n=9, 30±1 wks), women with idiopathic proteinuria (IP, n=15, 28±2 wks), PE mild features (mPE, n=16, 33±1 wks), superimposed PE (spPE, n=16, 31±1 wks), PE severe features (sPE, n=16, 32±1 wks) and HELLP (n=16, 29±1 wks). In the discovery phase, urine proteins were enriched by CR precipitation and subjected to UPLC-tandem mass spectrometry. Linear discriminant analysis (LDA) was used to cluster clinical groups based on molecular signatures. We choose a non-parsimonious set of peptides that distinguished among clinical phenotypes and were suitable for targeted validation using MRM in crude urine samples. 1,747 molecular IDs were identified in CR precipitates. These merged into 690 protein groups that generated good separation among clinical phenotypes with a false discovery rate of 1.12% (Fig. A). 21 key peptides were prioritized through pairwise comparison among groups (Wilkes Lambda MANOVA=0.006, p-value of 3.6 x 10-14) (Fig. B). Based on a full MS/MS spectral library, 98 MRM transitions were generated. CLIC1 (oxidant sensor activated by misfolded proteins), ABHD14B (lipid metabolizing-enzyme) and β2-microglobulin were among the parent proteins to distinguish sPE from HELLP. Principal component analysis demonstrated that targeted proteome from raw urine represented CR precipitate well and closely correlated with clinical criteria. Based on molecular signatures, HELLP syndrome appears to be a different clinical entity from sPE.

Helicobacter pylori Growth Stage Determines the Size, Protein Composition, and Preferential Cargo Packaging of Outer Membrane Vesicles.

Gram-negative bacteria release outer membrane vesicles (OMVs) as part of their normal growth that contain a range of cargo from their parent bacterium, including DNA, RNA, and proteins. The protein content of OMVs is suggested to be similar in composition to various sub-cellular locations of their parent bacterium. However, very little is known regarding the effect of bacterial growth stage on the size, content, and selective packaging of proteins into OMVs. In this study, the global proteome of Helicobacterpylori and their OMVs throughout bacterial growth are examined to determine if bacterial growth stage affected OMV cargo composition. Analysis of OMVs produced by H.pylori reveals that bacterial growth stage affects the size, composition, and selection of protein cargo into OMVs. Proteomic analysis identifies that the proteome of H.pylori OMVs is vastly different throughout bacterial growth and that OMVs contain a range of proteins compared to their parent bacteria. In addition, bacterial growth stage affects the ability of OMVs to induce the production of IL-8 by human epithelial cells. Therefore, the findings identify that the size, proteome, and immunogenicity of OMVs produced during various stages of bacterial growth is not comparable. Collectively, these findings highlight the importance of considering the bacterial growth stage from which OMVs are isolated, as this will impact their size, protein composition, and ultimately their biological functions.

Purification and functional characterization of a novel tyrosinase (diphenolase) inhibitory peptides prepared from Solunum tuberosum peels protein via enzymatic hydrolysis

Abstract The present study was designed to prepare a novel tyrosinase inhibitory peptides from Solunum tuberosum (potato) peels (by-product of potato processing). Isolated protein was hydrolyzed by immobilized lettuce protease at pH 10 to produce a potato hydrolysate (peptides) with the highest tyrosinase inhibitory activity than that of the parent protein. For maximum production of peptides, hydrolysis process including incubation temperature and protein concentration per reaction mixture was optimized. Separation of potato hydrolysate by ultrafiltration provides three fractions (F1, F2 and F3) of which F1 (

Expanding the RNA-Guided Endonuclease Toolkit for Mouse Genome Editing.

The RNA-guided endonuclease CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is widely used for generating genetically modified mice via zygotic microinjection. Although SpCas9 is a potent mutagen, it requires an NGG proto-spacer adjacent motif (PAM) at the target site, restricting sequence targetability. Here, we show that RNA-guided endonucleases that utilize a range of alternative PAM sequences can edit the mouse genome at the neurog3 (Ngn3) locus: SpCas9 VQR (NGAN PAM), SpCas9 VRER (NGCG), AsCas12a (TTTN), SaCas9 (NNGRRT), and SaCas9 KKH (NNNRRT). Additional experiments targeting tyrosinase and frizzled3 with SaCas9 KKH and its parent protein demonstrated that these endonucleases generated mutations in up to 100% of embryos across three loci. Remarkably, in contrast to wild-type SpCas9, these endonucleases frequently generated mutant embryos that retain unmodified alleles in both template-free and HDR-repair experiments. Our findings broaden PAM recognition options for mouse genome editing and identify SaCas9/SaCas9 KKH as useful alternatives when targeting genes with null lethal phenotypes.

Release of functional peptides from mother's milk and fortifier proteins in the premature infant stomach.

Digestion of milk proteins in the premature infant stomach releases functional peptides; however, which peptides are present has not been reported. Premature infants are often fed a combination of human milk and bovine milk fortifiers, but the variety of functional peptides released from both human and bovine milk proteins remains uncharacterized. This study applied peptidomics to investigate the peptides released in gastric digestion of mother’s milk proteins and supplemental bovine milk proteins in premature infants. Peptides were assessed for homology against a database of known functional peptides—Milk Bioactive Peptide Database. The peptidomic data were analyzed to interpret which proteases most likely released them from the parent protein. We identified 5,264 unique peptides from bovine and human milk proteins within human milk, fortifier or infant gastric samples. Plasmin was predicted to be the most active protease in milk, while pepsin or cathepsin D were predicted to be most active in the stomach. Alignment of the peptide distribution showed a different digestion pattern between human and bovine proteins. The number of peptides with high homology to known functional peptides (antimicrobial, angiotensin-converting enzyme-inhibitory, antioxidant, immunomodulatory, etc.) increased from milk to the premature infant stomach and was greater from bovine milk proteins than human milk proteins. The differential release of bioactive peptides from human and bovine milk proteins may impact overall health outcomes in premature infants.

ProteomicsBrowser: MS/proteomics data visualization and investigation.

Large-scale, quantitative proteomics data are being generated at ever increasing rates by high-throughput, mass spectrometry technologies. However, due to the complexity of these large datasets as well as the increasing numbers of post-translational modifications (PTMs) that are being identified, developing effective methods for proteomic visualization has been challenging. ProteomicsBrowser was designed to meet this need for comprehensive data visualization. Using peptide information files exported from mass spectrometry search engines or quantitative tools as input, the peptide sequences are aligned to an internal protein database such as UniProtKB. Each identified peptide ion including those with PTMs is then visualized along the parent protein in the Browser. A unique property of ProteomicsBrowser is the ability to combine overlapping peptides in different ways to focus analysis of sequence coverage, charge state or PTMs. ProteomicsBrowser includes other useful functions, such as a data filtering tool and basic statistical analyses to qualify quantitative data. ProteomicsBrowser is implemented in Java8 and is available at https://medicine.yale.edu/keck/nida/proteomicsbrowser.aspx and https://github.com/peng-gang/ProteomicsBrowser. Supplementary data are available at Bioinformatics online.

Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission.

Biosensors have become an indispensable tool set in life sciences. Among them, fluorescent protein-based biosensors have great biocompatibility and tunable emission properties but their development is largely on trial and error. To facilitate a rational design, we implement tunable femtosecond stimulated Raman spectroscopy, aided by transient absorption and quantum calculations, to elucidate the working mechanisms of a single-site Pro377Arg mutant of an emission ratiometric Ca2+ biosensor based on a green fluorescent protein-calmodulin complex. Comparisons with the parent protein and the Ca2+-free/bound states unveil more structural inhomogeneity yet an overall faster excited-state proton-transfer (ESPT) reaction inside the Ca2+-bound biosensor. The correlated photoreactant and photoproduct vibrational modes in the excited state reveal more chromophore twisting and trapping in the Ca2+-bound state during ESPT and the largely conserved chromophore dynamics in the Ca2+-free state from parent protein. The uncovered structural dynamics insights throughout an ESPT reaction inside a calcium biosensor provide important design principles in maintaining a hydrophilic, less compact, and more homogeneous environment with directional H-bonding (from the chromophore to surrounding protein residues) via bioengineering methods to improve the ESPT efficiency and quantum yield while maintaining photostability.

Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures.

The identification and quantification of proteins lags behind DNA sequencing methods in scale, sensitivity and dynamic range. Here we show that sparse amino acid sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescent labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor reductions in each molecule’s fluorescence following consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We demonstrate the method on synthetic and naturally-derived peptide molecules in zeptomole-scale quantities. We also fluorescently label phosphoserines and demonstrate single-molecule, positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should empower studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single molecule sequencing.

A12587 Urinary peptidomic biomarkers of renal function in heart transplant recipients

Objectives: Chronic kidney disease is common in patients after heart transplantation (HTx). We assessed whether in HTx recipients the proteomic urinary classifier CKD273 or sequenced urinary peptides revealing the parental proteins correlated with the estimated glomerular filtration rate (eGFR). Methods: In 368 HTx patients, we measured the urinary peptidome by capillary electrophoresis coupled with mass spectrometry and analyzed 48 urinary peptides with a detectable signal in > 95% of participants. After 9.1 months (median), all patients underwent a repeat assessment of eGFR and 351 (95.4%) had their urinary markers remeasured. Results: In multivariable Bonferroni-corrected analyses of the baseline data, a 1-SD increase in CKD273 was associated with a 11.4 (95% confidence interval, 7.25–15.5) ml/min/1.73 m2 lower eGFR and an odds ratio of 2.63 (1.56–4.46) for having eGFR < 60 ml/min/1.73 m2. While relating eGFR category at follow-up to baseline urinary biomarkers, CKD273 had higher (P = 0.007) area under the curve (0.75; CI, 0.70 to 0.80) than 24-h proteinuria (0.64; CI, 0.58 to 0.69). In partial least squares analysis, the strongest correlates of the multivariable-adjusted baseline eGFR were fragments of collagen I (positive) and the mucin-1 subunit α (inverse). Associations between the changes in eGFR and the urinary markers were inverse for CKD273 and mucin-1 and positive for urinary collagen I. Conclusion: In HTx patients, CKD273 is closer associated with renal dysfunction than 24-h proteinuria. Fragments of collagen I and mucin-1, respectively positively and inversely associated with eGFR and change in eGFR, are single-peptide markers associated with renal dysfunction.

The impact of slaughtering methods on physicochemical characterization of sheep myoglobin

Recent data showed that the consumption of halal meat is increasing around the world. However, little is known about the physiochemical and digestive properties of meat prepared by this method. The aim of this study was to assess the effects of enzymatic hydrolysis with digestive enzymes on sheep meat myoglobin (Mb) prepared by two different slaughtering methods: halal slaughter (HS) and industrial slaughter (IS). The texture profile analysis (TPA) of samples and their antioxidant activity, using a Trolox equivalent antioxidant capacity scale, were determined. Mb was selected for the confirmation of digestion tests. In addition, the production of reactive oxygen species (ROS) and heme degradation of Mb samples were determined using chemiluminescence and fluorescence spectroscopy, respectively. The results showed that IS-Mb had more hydrophobicity, produced more ROS, and had a greater tendency for aggregation compared with HS-Mb. Both higher aggregation and ROS production resulted in less digestion of IS-Mb. TPA tests showed an increase in tenderness of HS samples compared with IS samples. The extent of HS samples hydrolysis was significantly greater than IS samples when treated in parallel with pancreatic enzymes. Peptides generally have significantly higher antioxidant activity than their intact parental proteins. Thus, the high antioxidant activity of HS samples is consistent with their enhanced hydrolysis. Higher aggregation and ROS production both play a crucial role in less digestion of Non-HSMb.

Small Heat Shock Proteins, Amyloid Fibrils, and Nicotine Stimulate a Common Immune Suppressive Pathway with Implications for Future Therapies.

The α7 nicotinic acetylcholine receptor (α7nAChR) is central to the anti-inflammatory function of the vagus nerve in a physiological mechanism termed the inflammatory reflex. Studies on the inflammatory reflex have been instrumental for the current development of the field of bioelectronic medicine. An independent investigation of the biological role of αB-crystallin (HspB5), the most abundant gene transcript present in active multiple sclerosis lesions in human brains, also led to α7nAChR. Induction of experimental autoimmune encephalomyelitis (EAE) in HspB5-/- mice results in greater paralytic signs, increased levels of proinflammatory cytokines, and T-lymphocyte activation relative to wild-type animals. Administration of HspB5 was therapeutic in animal models of multiple sclerosis, retinal and cardiac ischemia, and stroke. Structure-activity studies established that residues 73-92 were as potent as the parent protein, but only when it formed amyloid fibrils. Amyloid fibrils and small heat shock proteins (sHsps) selectively bound α7nAChR on peritoneal macrophages (MΦs) and B lymphocytes, converting the MΦs to an immune suppressive phenotype and mobilizing the migration of both cell types from the peritoneum to secondary lymph organs. Here, we review multiple aspects of this work, which may be of interest for developing future therapeutic approaches for multiple sclerosis and other disorders.

Enhanced biofilm prevention activity of a SPLUNC1-derived antimicrobial peptide against Staphylococcus aureus.

SPLUNC1 is a multifunctional protein of the airway with antimicrobial properties. We previously reported that it displayed antibiofilm activities against P. aeruginosa. The goal of this study was to determine whether (1) the antibiofilm property is broad (including S. aureus, another prevalent organism in cystic fibrosis); (2) the α4 region is responsible for such activity; and (3), if so, this motif could be structurally optimized as an antimicrobial peptide with enhanced activities. We used S. aureus biofilm-prevention assays to determine bacterial biomass in the presence of SPLUNC1 and SPLUNC1Δα4 recombinant proteins, or SPLUNC1-derived peptides (α4 and α4M1), using the well-established crystal-violet biofilm detection assay. The SPLUNC1Δα4 showed markedly reduced biofilm prevention compared to the parent protein. Surprisingly, the 30-residue long α4 motif alone demonstrated minimal biofilm prevention activities. However, structural optimization of the α4 motif resulted in a modified peptide (α4M1) with significantly enhanced antibiofilm properties against methicillin–sensitive (MSSA) and–resistant (MRSA) S. aureus, including six different clinical strains of MRSA and the well-known USA300. Hemolytic activity was undetectable at up to 100μM for the peptides. The data warrant further investigation of α4-derived AMPs to explore the potential application of antimicrobial peptides to combat bacterial biofilm-related infections.

Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils

In the present study, whey protein nanofibrils (WPN) were used as carriers to improve the aqueous solubility of curcumin at acidic conditions (pH 3.2) for expanding its applications in functional beverages and drinks. Nanofibrils with nanometric diameter (less than 15 nm) and micrometric length were produced by 5 h heating (85 °C) of whey protein isolate (WPI) solution at pH 2.0. WPN showed a higher surface hydrophobicity compared to the native parental proteins which improved its ability to form a soluble complex with curcumin. The water solubility of curcumin was increased by about 1200-folds through the complexation with WPN, whereas the complexation with WPI increased its solubility by approximately 180-folds. Fluorescence measurements and Fourier transform infra-red spectroscopy indicated that the hydrogen bonding and hydrophobic interactions were mainly contributed to the formation of curcumin-WPN complexes. Circular dichroism spectroscopy revealed that the secondary structures of WPN were not significantly affected by binding to curcumin. The structural phase of nanocomplexes also was studied using X-ray diffraction analysis. Complexation of whey protein nanofibrils with curcumin improved their apparent viscosity and surface activity which makes them appropriate candidates to design new functional food emulsions and beverages. The results of antioxidant activity measurements (DPPH radical scavenging activity and reducing power) also showed that the antioxidant capacity of curcumin was drastically improved through the complexation with WPN. Generally, this study suggests that the whey protein nanofibril could be employed as a material to enhance the food applications of curcumin as a water-insoluble bioactive compound.

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.

Conformational Design and Characterisation of a Truncated Diamine Oxidase from Arthrobacter globiformis.

A functional mini protein can be developed by miniaturising its size. The minimisation technique provides an excellent model system for studying native enzymes, especially in creating an alternative novel biocatalyst. Miniaturised proteins may have enhanced stability, a crucial characteristic for large-scale production and industrial applications. In this study, a huge enzyme molecule, known as diamine oxidase (DAO, comprising 700 amino acids), was selected to undergo the process. By retaining the arrangement of the original functional sites of DAO in the fourth domain, a mini DAO can be designed via homology modelling. After several downsizing processes, a final configuration of 220 amino acids displayed high binding affinity towards histamine, a short-chain substrate that was catalysed by the parental DAO. The configuration also showed enhanced affinity towards a long-chain substrate known as spermidine. The gene for the designed protein was cloned and expressed in pET102/TOPO vector and overexpressed in E. coli BL21 (DE3). The new mini DAO had similar temperature tolerance and versatile substrates specificity characteristics as its parental protein. An active mini-protein with these characteristics is potentially useful for several applications such as detecting biogenic amines in the biological fluids and the environment that may give rise to health issues.

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochrome c.

The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (p Ka) of the protein. However, the reduction potential of the heme iron decreases; further, the p KH of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c.

Bioanalysis Rising Star Award 2018: announcing our finalists!

Bioanalysis Rising Star Award 2018: announcing our finalists!

Domain communication in Thermotoga maritima Arginine Binding Protein unraveled through protein dissection

Substrate binding proteins represent a large protein family that plays fundamental roles in selective transportation of metabolites across membrane. The function of these proteins relies on the relative motions of their two domains. Insights into domain communication in this class of proteins have been here collected using Thermotoga maritima Arginine Binding Protein (TmArgBP) as model system. TmArgBP was dissected into two domains (D1 and D2) that were exhaustively characterized using a repertoire of different experimental and computational techniques. Indeed, stability, crystalline structure, ability to recognize the arginine substrate, and dynamics of the two individual domains have been here studied. Present data demonstrate that, although in the parent protein both D1 and D2 cooperate for the arginine anchoring; only D1 is intrinsically able to bind the substrate. The implications of this finding on the mechanism of arginine binding and release by TmArgBP have been discussed. Interestingly, both D1 and D2 retain the remarkable thermal/chemical stability of the parent protein. The analysis of the structural and dynamic properties of TmArgBP and of the individual domains highlights possible routes of domain communication. Finally, this study generated two interesting molecular tools, the two stable isolated domains that could be used in future investigations.

Perspectives in the design of zein-based polymeric delivery systems with programmed wear down for sustainable agricultural applications

Design of nanostructured systems for pesticide delivery allows the implementation of a rational and sustainable use of these chemicals. Zein nanoparticles (ZNPs) enable extended foliar adhesion, as well as prolonged and controlled release of the entrapped agrochemical. The present study aimed to investigate degradation of ZNPs with various surface properties under environmentally relevant conditions. Accelerated degradation studies were performed on freeze-dried ZNPs synthesized with different cationic and non-ionic surfactants. These polymeric particles dispersed in alkaline media (pH = 9) revealed hydrodynamic diameters ranging between 210.6 and 297.3 nm and negative surface charges from −44.3 mV to −34.56 mV. Degradation profiles of the studied particles were assessed within the pH limits of soils under agricultural exploitation (pH 4 and 9). The time-dependent decrease of the parent protein fractions was monitored by capillary gel electrophoresis, where the rate constants of the hydrolytic degradation allows the estimation of ZNPs' persistence at any temperature of interest. Results showed that in alkaline media promotes a faster chemical degradation of ZNPs (total at 20 °C in ∼3918 days at pH = 4 vs. 205 days at pH = 9), however the nature of the used surfactant may completely reverse the observed trend. Identifying the technological variables that impact their surface chemistry and susceptibility towards hydrolytic degradation, opens new perspectives in the programmed degradation of ZNPs, which may ensure a safer and more sustainable use of these nanodelivery systems. Although chemometric models were able to assess quantitative correlations between recorded physical properties of the ZNPs and the estimated time necessary for their full degradation, further studies are needed to elucidate the causality between the nature of used surfactants and estimated degradation times in aqueous media.