Parent Protein Research Articles

OSTEO18, a novel urinary proteomic signature, associated with osteoporosis in heart transplant recipients

Abstract Background Immunosuppressive treatment in heart transplant (HTx) recipient causes osteoporosis. The urinary proteomic profile (UPP) includes peptide fragments derived from the bone extracellular matrix. Purpose Study aims were to develop and validate a multidimensional UPP biomarker for osteoporosis in HTx patients from single sequenced urinary peptides identifying the parent proteins. Methods A single-center HTx cohort was analyzed. Urine samples were measured by capillary electrophoresis coupled with mass spectrometry. Cases with osteoporosis and matching controls were randomly selected from all available 389 patients. In derivation case-control dataset, 1576 sequenced peptides detectable in ≥30% of patients. Applying statistical analysis on these, an 18-peptide multidimensional osteoporosis UPP biomarker (OSTEO18) was generated by support vector modeling. The 2 replication datasets included 118 and 94 patients. For further validation, the whole cohort was analyzed. Statistical methods included logistic regression and receiver operating characteristic curve (ROC) analysis. Results In derivation dataset, the AUC, sensitivity and specificity of OSTEO18 were 0.83 (95% CI: 0.76-0.90), 74.3% and 87.1%, respectively. In replication datasets, results were confirmatory. In the whole cohort (154 osteoporotic patients [39.6%]), the ORs for osteoporosis increased (p < 0.0001) across OSTEO18 quartiles from 0.39 (95% CI: 0.25-0.61) to 3.14 (2.08-4.75). With full adjustment for known osteoporosis risk factors, OSTEO18 improved AUC from 0.708 to 0.786 (p = 0.0003) for OSTEO18 categorized (optimized threshold: 0.095) and to 0.784 (p = 0.0004) for OSTEO18 as continuously distributed classifier. Conclusion OSTEO18 is a clinically meaningful novel biomarker indicative of osteoporosis in HTx recipients and is being certified as in-vitro diagnostic.

Solid-state fermentation of green lentils by Lactiplantibacillus plantarum leads to formation of distinct peptides that are absorbable and enhances DPP-IV inhibitory activity in an intestinal Caco-2 cell model.

Food-derived bioactive compounds mimicking the effects of incretin therapies offer promising opportunities for combination therapies with functional foods, where food matrix interactions, gastrointestinal enzyme activity, and in situ bioactivity should be key considerations. In this study, green lentils were solid-state fermented with Lactiplantibacillus plantarum ATCC8014, in vitro digested and exposed to brush border enzymes of a Caco-2 cell monolayer. Intestinal absorption of peptides and DPP-IV inhibitory activity were then investigated. LC-MS/MS profiles showed that peptides mainly originated from parental proteins of the vicilin, convicilin and legumin families. Fermentation led to the formation of more hydrophobic peptides when compared to the unfermented flour and up to 33.6% of them were transported to the basolateral side of a Caco-2 cell monolayer. Peptides with more than 22 amino acids and with a mass greater than 2000 Da were minimally transported. 73 peptides were uniquely identified in the basolateral fraction suggesting that they resulted from the activity of the brush border enzymes. The DPP-IV activity of Caco-2 cells grown as a polarized monolayer was decreased by 37.3% when exposed to in vitro digested 72 h-fermented lentil flour and 10% when exposed to the unfermented one. Inhibition of DPP-IV in the basolateral fluids was improved in a dose-dependent manner and reached 7.9% when 500 mg mL-1 of in vitro digested 72 h fermented lentil flour was used. Glucose absorption and uptake were minimally affected, suggesting that the previously observed hypoglycemic properties of lentils are likely due to activity on DPP-IV rather than on the inhibition of glucose absorption.

Early metabolic and hemodynamic indicators of kidney dysfunction in mice offspring from parental low protein diet

BackgroundParental malnutrition, particularly a low-protein diet (LPD), causes oligonephropathy at birth and predisposes offspring to hypertension and chronic kidney disease later in life. The onset of adult kidney disease varies based on genetics and environmental factors, often with subclinical alterations in kidney function being overlooked. This study aimed to examine changes in kidney morphology before significant kidney function decline in the offspring of mice fed a low-protein diet.MethodsUsing a combination of histological analysis, kidney metabolic and hemodynamic panel assessments, and advanced statistical techniques such as Linear Discriminant Analysis (LDA) and Principal Component Analysis (PCA), we investigated the initial impact of a maternal low-protein diet (LPD) on kidney development and function. Our study utilized 12-week-old F1 mice from F0 parents fed either a low-protein diet (LPD) or a normal-protein diet (NPD) before the onset of hypertension.ResultsThe offspring (F1 generation) of parents (F0 generation) fed an LPD show reduced body weight from birth to P20. The kidney weight was also reduced compared to F1 offspring from parents fed an NPD. At 12 weeks of age, body weight normalized, but kidney weight remained low. Offspring of parents fed an LPD displayed abnormal kidney morphology, including dilated tubules, oligonephropathy, and fluid-filled cysts which had worsened with age. A kidney metabolic panel analysis at 12 weeks revealed a slight but consistent increase in urine albumin, plasma creatinine, mean urea, and BUN concentrations. Although no significant changes in hemodynamic variables were observed, 2/12 mice, both males, showed alterations in systolic blood pressure, suggesting sex-specific effects when comparing F1 mice from F0 fed either diet. Overall, kidney metabolic changes were strongly correlated to parental LPD.ConclusionOur findings indicate that significant kidney damage must accumulate in the F1 generation from parents fed an LPD before any detectable changes in blood pressure occur. Our study suggests that small variations in kidney metabolic function may point to early kidney damage and should not be overlooked in the offspring of these malnourished mice and likely humans.

Nanodisc-associated acetylcholinesterase as a novel model system of physiological relevant membrane-bound cholinesterases. Inhibition by phenolic compounds

Acetylcholinesterase (AChE) plays a pivotal role in the cholinergic system, and its inhibition is sought after in a wide range of applications, from insect control to Alzheimer's disease treatment. While the primary physiological isoforms of AChE are membrane-bound proteins, most assays for discovering new, safer, and potent inhibitors are conducted using commercially available soluble isoforms, such as the electric eel AChE (eeAChE). In this study, we conducted a comparative analysis of the activity and selectivity to phenolic inhibitors of recombinant human AChE, eeAChE and a mutant variant of human AChE known as dAChE4. Despite numerous mutations, dAChE4 closely resembles its parental protein and serves as a suitable model for monomeric human AChE. We also established an in vitro system of membrane-bound AChE to create a model that closely mimics the physiological isoforms. This system ensures the proper work of the enzyme and allowed us to control the exact concentration of enzyme and lipids per assay.

Morphometric analysis of the intergenerational effects of protein restriction on nephron endowment in mice

BackgroundParental nutritional status is crucial in shaping offspring's kidney development. However, the association between a protein-restrictive diet and its intergenerational impact on kidney development remains unclear. MethodsWe conducted multigenerational morphometric measurements to investigate the effects of parental protein deprivation on offspring kidney development across four generations. F0 mice were divided into two groups and fed a normal protein diet (NPD) or a low-protein diet (LPD) for three weeks before mating and continued these diets throughout gestation and lactation. Body weight (BW), kidney weight (KW), KW/BW ratio, nephron counts, and blood pressure were assessed in F1 pups. To examine paternal effects, we bred CD1 females on an NPD with males on an LPD. BW, KW, KW/BW, and nephron counts were measured at P20. To measure the transgenerational effect of parental LPD on kidney development, F1 offspring (from parents on LPD) were fed NPD upon weaning. These F1 offspring were bred at 6 weeks of age to produce F2, F3 and F4 generations. Kidney metrics were evaluated across generations. ResultsThe average body weight of P0 pups from parents on NPD was 1.61g, while pups from parental LPD weighed an average of 0.869g, a decrease of 54 % (p = 6.9e-11, Wilcoxon test). F1 from parental LPD have significantly smaller kidneys than the control, with an average combined kidney weight of 0.0082g versus 0.0129g, a 37 % decrease (p = 3.2e-02, Wilcoxon test). P20 BW and KW remained low in LPD offspring. These effects persisted for 4 generations (F1 to F4) with an average glomerular count reduction of roughly 20 %. F3 and F4 showed wider variability in glomerular counts but were not statistically significant compared to controls. ConclusionsBoth maternal and paternal LPD significantly affected offspring nephron endowment. Our study underscores the complex nature of nutritional transgenerational effects on kidney development, emphasizing the importance of both maternal and paternal dietary impacts on kidney development and the developmental origin of adult disease.

CircSSR1 regulates pyroptosis of pulmonary artery smooth muscle cells through parental protein SSR1 mediating endoplasmic reticulum stress

IntroductionPyroptosis, inflammatory necrosis of cells, is a programmed cell death involved in the pathological process of diseases. Endoplasmic reticulum stress (ERS), as a protective stress response of cell, decreases the unfold protein concentration to inhibit the unfold protein agglutination. Whereas the relationship between endoplasmic reticulum stress and pyroptosis in pulmonary hypertension (PH) remain unknown. Previous evident indicated that circular RNA (circRNA) can participate in several biological process, including cell pyroptosis. However, the mechanism of circRNA regulate pyroptosis of pulmonary artery smooth muscle cells through endoplasmic reticulum stress still unclear. Here, we proved that circSSR1 was down-regulate expression during hypoxia in pulmonary artery smooth muscle cells, and over-expression of circSSR1 inhibit pyroptosis both in vitro and in vivo under hypoxic. Our experiments have indicated that circSSR1 could promote host gene SSR1 translation via m6A to activate ERS leading to pulmonary artery smooth muscle cell pyroptosis. In addition, our results showed that G3BP1 as upstream regulator mediate the expression of circSSR1 under hypoxia. These results highlight a new regulatory mechanism for pyroptosis and provide a potential therapy target for pulmonary hypertension.MethodsRNA-FISH and qRT-PCR were showed the location of circSSR1 and expression change. RNA pull-down and RIP verify the circSSR1 combine with YTHDF1. Western blotting, PI staining and LDH release were used to explore the role of circSSR1 in PASMCs pyroptosis.ResultsCircSSR1 was markedly downregulated in hypoxic PASMCs. Knockdown CircSSR1 inhibited hypoxia induced PASMCs pyroptosis in vivo and in vitro. Mechanistically, circSSR1 combine with YTHDF1 to promote SSR1 protein translation rely on m6A, activating pyroptosis via endoplasmic reticulum stress. Furthermore, G3BP1 induce circSSR1 degradation under hypoxic.ConclusionOur findings clarify the role of circSSR1 up-regulated parental protein SSR1 expression mediate endoplasmic reticulum stress leading to pyroptosis in PASMCs, ultimately promoting the development of pulmonary hypertension.

Urinary proteomic signature of mineralocorticoid receptor antagonism by spironolactone: evidence from the HOMAGE trial

ObjectiveHeart failure (HF) is characterised by collagen deposition. Urinary proteomic profiling (UPP) followed by peptide sequencing identifies parental proteins, for over 70% derived from collagens. This study aimed to refine...

Using established biorepositories for emerging research questions: a feasibility study

BackgroundProteomics and metabolomics offer substantial potential for advancing kidney transplant research by providing versatile opportunities for gaining insights into the biomolecular processes occurring in donors, recipients, and grafts. To achieve this, adequate quality and numbers of biological samples are required. Whilst access to donor samples is facilitated by initiatives such as the QUOD biobank, an adequately powered biobank allowing exploration of recipient-related aspects in long-term transplant outcomes is missing. Rich, yet unverified resources of recipient material are the serum repositories present in the immunological laboratories of kidney transplant centers that prospectively collect recipient sera for immunological monitoring. However, it is yet unsure whether these samples are also suitable for -omics applications, since such clinical samples are collected and stored by individual centers using non-uniform protocols and undergo an undocumented number of freeze–thaw cycles. Whilst these handling and storage aspects may affect individual proteins and metabolites, it was reasoned that incidental handling/storage artifacts will have a limited effect on a theoretical network (pathway) analysis. To test the potential of such long-term stored clinical serum samples for pathway profiling, we submitted these samples to discovery proteomics and metabolomics.MethodsA mass spectrometry-based shotgun discovery approach was used to obtain an overview of proteins and metabolites in clinical serum samples from the immunological laboratories of the Dutch PROCARE consortium. Parallel analyses were performed with material from the strictly protocolized QUOD biobank.ResultsFollowing metabolomics, more than 800 compounds could be identified in both sample groups, of which 163 endogenous metabolites were found in samples from both biorepositories. Proteomics yielded more than 600 proteins in both groups. Despite the higher prevalence of fragments in the clinical, non-uniformly collected samples compared to the biobanked ones (42.5% vs 26.5% of their proteomes, respectively), these fragments could still be connected to their parent proteins. Next, the proteomic and metabolomic profiles were successfully mapped onto theoretical pathways through integrated pathway analysis, which showed significant enrichment of 79 pathways.ConclusionsThis feasibility study demonstrated that long-term stored serum samples from clinical biorepositories can be used for qualitative proteomic and metabolomic pathway analysis, a notion with far-reaching implications for all biomedical, long-term outcome-dependent research questions and studies focusing on rare events.

A plasma peptidomic signature reveals extracellular matrix remodeling and predicts prognosis in alcohol-associated hepatitis.

Alcohol-associated hepatitis (AH) is plagued with high mortality and difficulty in identifying at-risk patients. The extracellular matrix undergoes significant remodeling during inflammatory liver injury and could potentially be used for mortality prediction. EDTA plasma samples were collected from patients with AH (n = 62); Model for End-Stage Liver Disease score defined AH severity as moderate (12-20; n = 28) and severe (>20; n = 34). The peptidome data were collected by high resolution, high mass accuracy UPLC-MS. Univariate and multivariate analyses identified differentially abundant peptides, which were used for Gene Ontology, parent protein matrisomal composition, and protease involvement. Machine-learning methods were used to develop mortality predictors. Analysis of plasma peptides from patients with AH and healthy controls identified over 1600 significant peptide features corresponding to 130 proteins. These were enriched for extracellular matrix fragments in AH samples, likely related to the turnover of hepatic-derived proteins. Analysis of moderate versus severe AH peptidomes was dominated by changes in peptides from collagen 1A1 and fibrinogen A proteins. The dominant proteases for the AH peptidome spectrum appear to be CAPN1 and MMP12. Causal graphical modeling identified 3 peptides directly linked to 90-day mortality in >90% of the learned graphs. These peptides improved the accuracy of mortality prediction over the Model for End-Stage Liver Disease score and were used to create a clinically applicable mortality prediction assay. A signature based on plasma peptidome is a novel, noninvasive method for prognosis stratification in patients with AH. Our results could also lead to new mechanistic and/or surrogate biomarkers to identify new AH mechanisms.

Characterization of maltose-binding protein-fused heparinases with enhanced thermostability by application of rigid and flexible linkers.

Heparinases, including heparinases I-III (HepI, HepII, and HepIII, respectively), are important tools for producing low-molecular-weight heparin, an improved anticoagulant. The poor thermostability of heparinases significantly hinders their industrial and laboratory applications. To improve the thermostability of heparinases, we applied a rigid linker (EAAAK)5 (R) and a flexible linker (GGGGS)5 (F) to fuse maltose-binding protein (MBP) and HepI, HepII, and HepIII from Pedobacter heparinus, replacing the original linker from the plasmid pMAL-c2X. Compared with their parental fusion protein, MBP-fused HepIs, HepIIs, and HepIIIs with linkers (EAAAK)5 or (GGGGS)5 all displayed enhanced thermostability (half-lives at 30°C: 242%-464%). MBP-fused HepIs and HepIIs exhibited higher specific activity (127%-324%), whereas MBP-fused HepIIIs displayed activity similar to that of their parental fusion protein. Kinetics analysis revealed that MBP-fused HepIIs showed a significantly decreased affinity toward heparin with increased Km values (397%-480%) after the linker replacement, whereas the substrate affinity did not change significantly for MBP-fused HepIs and HepIIIs. Furthermore, it preliminarily appeared that the depolymerization mechanism of these fusion proteins may not change after linker replacement. These findings suggest the superior enzymatic properties of MBP-fused heparinases with suitable linker designs and their potential for the bioproduction of low-molecular-weight heparin.

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

A multicenter, randomized, double-blind, placebo-controlled ascending dose study to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamic (PD) effects of Posiphen in subjects with early Alzheimer’s Disease

BackgroundAmyloid beta protein (Aβ) is a treatment target in Alzheimer’s Disease (AD). Lowering production of its parent protein, APP, has benefits in preclinical models. Posiphen, an orally administered small molecule, binds to an iron-responsive element in APP mRNA and decreases translation of APP and Aβ. To augment human data for Posiphen, we evaluated safety, tolerability and pharmacokinetic and pharmacodynamic (PD) effects on Aβ metabolism using Stable Isotope Labeling Kinetic (SILK) analysis.MethodsDouble-blind phase 1b randomized ascending dose clinical trial, at five sites, under an IRB-approved protocol. Participants with mild cognitive impairment or mild AD (Early AD) confirmed by low CSF Aβ42/40 were randomized (within each dose arm) to Posiphen or placebo. Pretreatment assessment included lumbar puncture for CSF. Participants took Posiphen or placebo for 21–23 days, then underwent CSF catheter placement, intravenous infusion of 13C6-leucine, and CSF sampling for 36 h. Safety and tolerability were assessed through participant reports, EKG and laboratory tests. CSF SILK analysis measured Aβ40, 38 and 42 with immunoprecipitation-mass spectrometry. Baseline and day 21 CSF APP, Aβ and other biomarkers were measured with immunoassays. The Mini-Mental State Exam and ADAS-cog12 were given at baseline and day 21.ResultsFrom June 2017 to December 2021, 19 participants were enrolled, randomized within dose cohorts (5 active: 3 placebo) of 60 mg once/day and 60 mg twice/day; 1 participant was enrolled and completed 60 mg three times/day. 10 active drug and 5 placebo participants completed all study procedures. Posiphen was safe and well-tolerated. 8 participants had headaches related to CSF catheterization; 5 needed blood patches. Prespecified SILK analyses of Fractional Synthesis Rate (FSR) for CSF Aβ40 showed no significant overall or dose-dependent effects of Posiphen vs. placebo. Comprehensive multiparameter modeling of APP kinetics supported dose-dependent lowering of APP production by Posiphen. Cognitive measures and CSF biomarkers did not change significantly from baseline to 21 days in Posiphen vs. placebo groups.ConclusionsPosiphen was safe and well-tolerated in Early AD. A multicenter SILK study was feasible. Findings are limited by small sample size but provide additional supportive safety and PK data. Comprehensive modeling of biomarker dynamics using SILK data may reveal subtle drug effects.Trial registrationNCT02925650 on clinicaltrials.gov (registered on 10-24-2016).

Stem Cell-Based Acellular Therapy: Insight into Biogenesis, Bioengineering and Therapeutic Applications of Exosomes.

The vast regenerative potential of stem cells has laid the foundation for stem cell-based therapies. However, certain challenges limit the application of cell-based therapies. The therapeutic use of cell-free therapy can avoid limitations associated with cell-based therapies. Acellular stem cell-based therapies rely on the use of biological factors released by stem cells, including growth factors and extracellular vesicles such as exosomes. Due to their comparable regenerative potential, acellular therapies may provide a feasible and scalable alternative to stem cell-based therapies. Exosomes are small vesicles secreted by various types of cells, including stem cells. Exosomes contain parent cell-derived nucleic acids, proteins, lipids, and other bioactive molecules. They play an important role in intra-cellular communication and influence the biological characteristics of cells. Exosomes inherit the properties of their parent cells; therefore, stem cell-derived exosomes are of particular interest for applications of regenerative medicine. In comparison to stem cell-based therapy, exosome therapy offers several benefits, such as easy transport and storage, no risk of immunological rejection, and few ethical dilemmas. Unlike stem cells, exosomes can be lyophilized and stored off-the-shelf, making acellular therapies standardized and more accessible while reducing overall treatment costs. Exosome-based acellular treatments are therefore readily available for applications in patients at the time of care. The current review discusses the use of exosomes as an acellular therapy. The review explores the molecular mechanism of exosome biogenesis, various methods for exosome isolation, and characterization. In addition, the latest advancements in bioengineering techniques to enhance exosome potential for acellular therapies have been discussed. The challenges in the use of exosomes as well as their diverse applications for the diagnosis and treatment of diseases have been reviewed in detail.

Colloidal and interfacial properties of spray dried pulse protein-blueberry polyphenol particles in model dispersion systems

The phytochemical composition and physicochemical attributes of polyphenol-enriched protein particle ingredients produced with pulse proteins (e.g. chickpea protein, pea protein, and a chickpea-pea protein blend) and polyphenols recovered from wild blueberry pomace were investigated for colloidal and interfacial properties. Anthocyanins were the major polyphenol fraction (27.74–36.47 mg C3G/g) of these polyphenol-rich particles (44.95–62.08 mg GAE/g). Dispersions of pea protein-polyphenol particles showed a superior phase stability before and after heat treatment compared to the chickpea pea protein-polyphenol system. This observation was independent of the added amount of NaCl in the dispersion. In general, at quasi equilibrium state, pulse protein-polyphenol particles and parental pulse protein ingredients showed similar oil-water interfacial tension. However, pea protein-polyphenol particles demonstrated a reduced diffusion-driven oil-water interfacial adsorption rate constant compared to the parental pea protein ingredient. Overall, the obtained results suggest application potential of pea protein-polyphenol particles as a functional food/beverage ingredient.

Production of Antioxidant Peptides from Snakehead Fish Using Batch and Continuous Enzymatic Hydrolysis

Bioactive peptides are promising functional ingredients. Due to its high protein content, snakehead fish (Channa striata) extract (SHFE) is one of the suitable parent proteins for bioactive peptides. This study aimed to investigate the production of SHFE-based antioxidative peptides in a conventional batch and continuous system facilitated by an enzymatic membrane reactor (EMR). The effects of different proteases (Alcalase, Neutrase), substrate concentrations, and enzyme-to-substrate ratios were investigated in the batch process. Continuous hydrolysis was then performed under the optimum conditions obtained from the batch process. The optimum conditions based on the antioxidant capacity measured by DPPH and FRAP assays were employing Alcalase with a substrate concentration [S] of 3% (w/v) and an enzyme-to-substrate ratio [E]/[S] of 10% (w/w). Continuous operation was shown to have been performed over a prolonged period, based on the calculated fouling rate. Furthermore, filtration of the resulting permeate with a smaller membrane pore size (2-kDa) increased the antioxidant capacity. This study is expected to increase the production of functional ingredients in snakehead fish.

Intestinal health of squab pigeons responded to parental dietary protein levels during breeding period

The objective of this study was to determine the effects of dietary crude protein (CP) levels on intestinal antioxidant status, tight junction proteins expression, and amino acids transporters levels in squabs. A total of 180 pairs of White King parent pigeons approximately 10 mo old were randomly assigned to 5 groups with 6 replications of 6 pairs of parental pigeons each, and were fed with 14, 15, 16, 17, and 18% CP diets for 46 d, respectively. Dietary increasing CP levels increased final body weight (linear and quadratic, P < 0.05), serum urea nitrogen (linear, P<0.05) and triglyceride levels (quadratic, P < 0.05), and reduced kidney relative weight (quadratic, P < 0.05) in squabs. Final body weight of squabs in the 18% CP diet group was higher than that of the 14, 15, and 16% CP diet groups (P < 0.05) but was similar to that of the 17% CP diet group (P > 0.05). Increasing dietary CP levels reduced intestinal malondialdehyde contents (linear and quadratic, P < 0.05) and jejunal total superoxide dismutase (T-SOD) activity (linear, P < 0.05), and enhanced (linear and quadratic, P<0.05) ileal catalase and T-SOD activities in squabs, and these effects were more prominent in the 17% CP diet group. Graded CP levels up-regulated the mRNA expression of intestinal zonula occludens 1 (linear, P < 0.05), solute carrier family 7 members 9 (linear, P < 0.05) and claudin 1 (CLDN1, linear and quadratic, P < 0.05), ileal CLDN3 and solute carrier family 6 members 14 (linear, P < 0.05) but lowered jejunal solute carrier family 6 member 14 (quadratic, P<0.05) mRNA expression in squabs. The effects of dietary CP levels on intestinal tight junction proteins expression were more apparent when its supplemental levels were 18%. These results suggested that increasing parental dietary CP levels ranged from 14 to 18% during breeding period improved growth and intestinal function of squabs, with its recommended level being 17%.

The physical and evolutionary energy landscapes of devolved protein sequences corresponding to pseudogenes

Protein evolution is guided by structural, functional, and dynamical constraints ensuring organismal viability. Pseudogenes are genomic sequences identified in many eukaryotes that lack translational activity due to sequence degradation and thus over time have undergone "devolution." Previously pseudogenized genes sometimes regain their protein-coding function, suggesting they may still encode robust folding energy landscapes despite multiple mutations. We study both the physical folding landscapes of protein sequences corresponding to human pseudogenes using the Associative Memory, Water Mediated, Structure and Energy Model, and the evolutionary energy landscapes obtained using direct coupling analysis (DCA) on their parent protein families. We found that generally mutations that have occurred in pseudogene sequences have disrupted their native global network of stabilizing residue interactions, making it harder for them to fold if they were translated. In some cases, however, energetic frustration has apparently decreased when the functional constraints were removed. We analyzed this unexpected situation for Cyclophilin A, Profilin-1, and Small Ubiquitin-like Modifier 2 Protein. Our analysis reveals that when such mutations in the pseudogene ultimately stabilize folding, at the same time, they likely alter the pseudogenes' former biological activity, as estimated by DCA. We localize most of these stabilizing mutations generally to normally frustrated regions required for binding to other partners.

Anti-bacterial compounds from Indian curry-leaf tree Murraya koenigii have potential to inhibit carbapenem-resistant Streptococcus pneumoniae

Problems consideredSpike in the mortality rates due to pneumococcal infections post-COVID-19 has raised clinical concerns globally. β-lactam antibiotic-susceptibility in the causative organism, Streptococcus pneumoniae has been compromised due to non-synonymous single nucleotide polymorphisms (nsSNPs) in different penicillin-binding protein (PBP) targets. Carbapenem-resistant S. pneumoniae strains have further constricted the pharmacologically safe therapeutic options. MethodsIn-house sequenced whole genomes of meropenem and cefotaxime resistant Indian and global isolates were used to retrieve parent and mutant proteins. Anti-bacterial phtytochemicals (ABPs) from Indian curry-leaf tree Murraya koenigii were identified using database-mining. ABPs were screeened based on pharmacokinetics, and toxicity profiles. Site-specific molecular docking studies revealed ABPs‘ affinity towards the targets as compared to control drug meropenem. Coarse-grained and normal-mode molecular dynamics simulation further asserted the interactive stability of the chosen compounds. ResultsFrom the whole genomes (n = 28) of meropenem and cefotaxime resistant (MIC ≥0.5 μg/ml) Indian and global isolates, ∼50 nsSNPs in the active-site regions of PBP1A, PBP2B and PBP2X were identified. Based on pharmacological safety and high drug-likeliness, 10 ABPs were funnelled down from 226 ABPs in M. koenigii. A pyrano-carbazole alkaloid koenine was further prioritized as the most efficient candidate based on consistently high binding affinity (binding energies −7.7 to −8.9 kcal/mol), low inhibition constant (0.69–3.5 μM) and low structural fluctuations (<1.1 Å), against mutant targets as compared to meropenem. ConclusionThe study encourages experimental validation of koenine against pneumococci and also promotes the use of similar phytochemical resources for evaluation against emerging drug-resistant pathogens harbouring mutant targets.

Fragment ion intensity prediction improves the identification rate of non-tryptic peptides in timsTOF

Immunopeptidomics is crucial for immunotherapy and vaccine development. Because the generation of immunopeptides from their parent proteins does not adhere to clear-cut rules, rather than being able to use known digestion patterns, every possible protein subsequence within human leukocyte antigen (HLA) class-specific length restrictions needs to be considered during sequence database searching. This leads to an inflation of the search space and results in lower spectrum annotation rates. Peptide-spectrum match (PSM) rescoring is a powerful enhancement of standard searching that boosts the spectrum annotation performance. We analyze 302,105 unique synthesized non-tryptic peptides from the ProteomeTools project on a timsTOF-Pro to generate a ground-truth dataset containing 93,227 MS/MS spectra of 74,847 unique peptides, that is used to fine-tune the deep learning-based fragment ion intensity prediction model Prosit. We demonstrate up to 3-fold improvement in the identification of immunopeptides, as well as increased detection of immunopeptides from low input samples.

Sulfoxide‐Mediated Cys‐Trp‐Selective Bioconjugation that Enables Protein Labeling and Peptide Heterodimerization

AbstractA method was developed that enables the magnesium chloride (MgCl2)‐activated S‐acetamidomethyl cysteine sulfoxide (Cys(Acm)(O)) to induce the sp2(C−H) sulfenylation of the indole of Trp residues. The reaction operates under mild acidic conditions using acetic acid or an ionic liquid to give the Trp‐sulfenylated products. Other than Trp, all other proteinogenic amino acids are unreactive to the sulfenylation conditions. We demonstrated the successful application of this reaction to various peptides, including lysozyme. Furthermore, we achieved the Trp‐modification of a monoclonal antibody by a MgCl2‐mediated reaction in an acidic ionic liquid. The resulting antibody exhibited antibody performance comparable to the parent protein. The amide moiety in the Acm group contributes to the difference in chemical behaviors between S‐Acm and S‐p‐methoxybenzyl (MBzl)‐protected cysteine sulfoxides. This is because the S‐Acm sulfoxide is converted to S‐chlorocysteine responsible for Trp‐sulfenylation under less acidic conditions than those required for the reaction of S‐MBzl sulfoxide. Based on this rationale, we prepared a linker possessing S‐Acm and S‐MBzl oxide moieties and subjected the linker to heterodimerization of DNA‐binding MYC and MAX peptides containing a Trp handle. The one‐pot/stepwise Cys‐Trp conjugation between the linker and DNA‐binding peptides allowed the generation of a heterodimeric MYC/MAX DNA binder.