Protein Degradation Products Research Articles

Astral-based DIA proteomics explored the flavor enhancement mechanism of Chinese traditional smoked bacon by staphylococcal co-fermentation

The proteolysis pattern during mixed fermentation of Staphylococcus cohnii WX-M8 and S. saprophyticus MY-A10 on Chinese bacon was still unknown. In this study, the changing laws of protein degradation products during staphylococcal mixed fermentation were analyzed, followed by an investigation of endogenous enzymes and cellular components, and finally an examination of flavor profiles. Results indicated that mixed fermentation improved protein degradation and promoted the production of peptides and free amino acids (FAAs). Proteolysis of S. saprophyticus MY-A10 was non-specific, and it promoted protein degradation by cooperating with cathepsin L1. S. cohnii WX-M8 was specific and acted mainly with calpain-3 in the thin filament. The fulfillment of S. cohnii WX-M8 function was enhanced in the presence of S. saprophyticus MY-A10. Mixed fermentation showed synergism with endogenous peptidases in degrading peptides to small-molecule peptides or FAAs and complementarity with endogenous dehydrogenases in converting FAAs to volatile organic compounds (VOCs).

Abstract Mo118: Myofilament proteolysis may underlie contractile remodeling in atrial fibrillation

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function, including decreased blood flow velocity and reduced atrial contractility, known as atrial stunning, occurs. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. In atrial cardiomyocytes that were useable for skinned single cardiomyocyte force-calcium studies, we found reduced force of contraction. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, MHC, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed increased calcium sensitivity that was consistent with increased cTnI degradation and decreased TnI phosphorylation. These results provide an understanding of the contractile remodeling that occurs in AFib.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared with sinus rhythm atria. Atrial cardiomyocytes show reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and troponin I altering calcium sensitivity. We measured degradation of other myofilament proteins, including cardiac myosin binding protein C and actinin, that show degradation products in the AFib samples that are absent in the sinus rhythm atria. Many of the degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.NEW & NOTEWORTHY Atrial fibrillation is the most common cardiac rhythm disorder, and remodeling during atrial fibrillation is highly variable between patients. This study has defined the biophysical changes in contractility that occur in atrial fibrillation along with identifying potential molecular mechanisms that may drive this remodeling. This includes proteolysis of several myofilament proteins including titin, troponin I, myosin heavy chain, myosin binding protein C, and actinin, which is consistent with the observed contractile deficits.

Intravenous injection of nattokinase-heparin electrostatic complex improves the therapeutic effect of advanced tumors by dissolving cancer-related thrombosis

AimsCancer-related thrombosis (CAT) is a common complication in cancer patients, significantly impacting their quality of life and survival prospects. Nattokinase (NK) has potent thrombolytic properties, however, its efficacy is limited by low oral bioavailability and the risk of severe allergic reactions with intravenous use. Heparin (HP) is a widely used anticoagulant in clinical settings. This study aimed to overcome the intravenous toxicity of NK and explore its effect on CAT in advanced tumors. Main methodsIn this study, NK-HP electrostatic complexes were constructed, and their safety and thrombolytic efficacy were verified through guinea pig allergy tests, mouse tail vein tests, and both in vivo and in vitro thrombolysis experiments. Additionally, an S180 advanced tumor model was developed and combined with sialic acid-modified doxorubicin liposomes (DOX-SAL) to investigate the impact of NK-HP on CAT and its antitumor effects in advanced tumors. Key findingsWe observed that NK-HP can eliminate the intravenous injection toxicity of NK, has strong thrombolytic performance, and can prevent thrombosis formation. Intravenous injection of NK-HP can enhance the antitumor effect of DOX-SAL by reducing the fibrin content in advanced tumors and increasing the levels of the cross-linked protein degradation product D-dimer. SignificanceThis study developed a method to eliminate the intravenous injection toxicity of NK, proposing a promising therapeutic strategy for CAT treatment, particularly for CAT in advanced tumors, and improving the efficacy of nano-formulations in anti-tumor therapy.

Increasing the Survival of a Neuronal Model of Alzheimer's Disease Using Docosahexaenoic Acid, Restoring Endolysosomal Functioning by Modifying the Interactions between the Membrane Proteins C99 and Rab5.

Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer's disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.

Evaluation of beneficial effects of dexpanthenol on hypoxic-ischemic encephalopathy

ABSTRACT Hypoxic-ischemic encephalopathy (HIE) is a cause of serious morbidity and mortality in newborns. Dexpanthenol, which is metabolized into D-pantothenic acid, has antioxidant and other potentially therapeutic properties. We examined some effects of dexpanthenol on the brains of week-old rat pups with HIE induced by obstruction of the right carotid artery followed by keeping in 8% O2 for 2 hours. Dexpanthenol (500 mg/kg) was administered intraperitoneally to 16 of 32 pups with HIE. Protein, DNA, and lipid oxidation degradation products were assayed and hippocampal and cortical cell apoptosis and neuronal cell numbers were evaluated in stained sections. Dexpanthenol application reduced oxidative stress and inflammation. TNF-α and IL-6 cytokine levels in HIE also decreased with dexpanthenol treatment. The numbers of caspase-3 positive cells in the dentate gyrus and CA1/CA2/CA3 regions of the hippocampus was lower, and apoptosis was decreased in the dexpanthenol-treated animals. These findings suggest possible clinical applications of dexpanthenol in human HIE.

A Crucial Role of Proteolysis in the Formation of Intracellular Dinitrosyl Iron Complexes.

Dinitrosyl iron complexes (DNICs) stabilize nitric oxide in cells and tissues and constitute an important form of its storage and transportation. DNICs may comprise low-molecular-weight ligands, e.g., thiols, imidazole groups in chemical compounds with low molecular weight (LMWDNICs), or high-molecular-weight ligands, e.g., peptides or proteins (HMWDNICs). The aim of this study was to investigate the role of low- and high-molecular-weight ligands in DNIC formation. Lysosomal and proteasomal proteolysis was inhibited by specific inhibitors. Experiments were conducted on human erythroid K562 cells and on K562 cells overexpressing a heavy chain of ferritin. Cell cultures were treated with •NO donor. DNIC formation was monitored by electron paramagnetic resonance. Pretreatment of cells with proteolysis inhibitors diminished the intensity and changed the shape of the DNIC-specific EPR signal in a treatment time-dependent manner. The level of DNIC formation was significantly influenced by the presence of protein degradation products. Interestingly, formation of HMWDNICs depended on the availability of LMWDNICs. The extent of glutathione involvement in the in vivo formation of DNICs is minor yet noticeable, aligning with our prior research findings.

Derlin-1 promotes diet-induced non-alcoholic fatty liver disease via increasing RIPK3-mediated necroptosis

Background & aimsUnrestricted endoplasmic reticulum (ER) stress and the continuous activation of ER associated protein degradation (ERAD) pathway might lead to the aggravation of non-alcoholic steatohepatitis (NASH). Derlin-1 has been considered to be an integral part of the ERAD pathway, which is involved in the regulation of the transport and excretion of protein degradation products within ER. However, the regulatory role and mechanism of Derlin-1 in NASH remains unclear. MethodsThe expression of Derlin-1 was firstly detected in the liver of normal and NASH animal model and patient. Then, western diet (WD)-induced NASH mice were administrated with the lentivirus-mediated Derlin-1 knockdown or overexpression. Finally, RIPK3 knockout mice were used to explore the mechanism. The liver injury, hepatic steatosis, inflammation, and fibrosis as well as ER stress signal pathway were evaluated. ResultsThe levels of Derlin-1 were significantly elevated in the liver of WD-fed mice and NASH patients when compared to the control group. Furthermore, Derlin-1 knockdown attenuated WD-induced liver injury, lipid accumulation, inflammatory response, and fibrosis. Conversely, overexpression of Derlin-1 presented the completely opposite results. Mechanistically, Derlin-1 enhanced ER stress pathways and led to necroptosis, and RIPK3 knockout dramatically reduced Derlin-1 expression and reversed the progression of NASH aggravated by Derlin-1. ConclusionsNotably, Derlin-1 is a critical modulator in NASH. It may accelerate the progression of NASH by regulating the activation of the ERAD pathway and further aggravating the ER stress, which might be involved in RIPK3-mediated necroptosis. Therefore, targeting Derlin-1 as a novel intervention point holds the potential to delay or even reverse NASH.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits.

Two Different Strategies for Stabilization of Brain Tissue and Extraction of Neuropeptides.

Neuropeptides are bioactive peptides that are synthesized and secreted by neurons in signaling pathways in the brain. Peptides and proteins are extremely vulnerable to proteolytic cleavage when their biological surrounding changes. This makes neuropeptidomics challenging due to the rapid alterations that occur to the peptidome after harvesting of brain tissue samples. For a successful neuropeptidomic study, the biological tissue sample analyzed should resemble the living state as much as possible. Heat stabilization has been proven to inhibit postmortem degradation by denaturing proteolytic enzymes, hence increasing identification rates of neuropeptides. Here, we describe two different stabilization protocols for rodent brain samples that increase the number of intact mature neuropeptides and minimize interference from degradation products of abundant proteins. Additionally, we present an extraction protocol that aims to extract a wide range of hydrophilic and hydrophobic neuropeptides by sequentially using an aqueous and an organic extraction medium.

High-sensitivity profiling of dipeptides in sauce-flavor Baijiu Daqu by chemical derivatization and ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry

Dipeptides in sauce-flavor Baijiu Daqu are protein degradation products during the fermentation of Daqu, which are believed to play a crucial role in the flavor and quality of Baijiu. Herein, we integrated dansyl chloride derivatization with ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) for comprehensively profiling dipeptides in Daqu. The derivatization efficiency was higher than 99.1 % for all 17 dipeptide standards under the optimized derivatization conditions. In total, 118 dipeptides were detected in Daqu. The method was validated and the analytical characteristics including the linearity (spanned across 2–4 orders of magnitude), precision (1.2–19.9 %), limit of detection (varied from 1.1 to 53.4 pmol/mL) and the stability (3.6–15.8 %) are satisfactory. The usefulness of the method was examined by studying the distribution characteristics of dipeptides in Daqu under different production conditions. The present method provides an effective and robust strategy for comprehensively analyzing dipeptide compounds in complex biological samples.

Catalytic Pyrolysis of the Green Microalgae Botryococcus braunii over Ni/SBA-15 Prepared by the Ultrasonic-Assisted Sol-Gel Method.

The pyrolysis of the green microalgae Botryococcus braunii in the absence and the presence of Ni/SBA-15 prepared by the ultrasonic-assisted sol-gel was investigated using pyrolysis-gas chromatography-mass spectroscopy (Py-GC/MS). Pyrolysis experiments were performed at 350, 450, and 550 °C under helium (He) flow. In the absence of a catalyst, the chemical composition of pyrolysis products at different temperatures, based on the relative peak area, comprised protein/amino acid derivative products of 9-15%, carbohydrate derivative products of 5-10%, lipid derivative products of 13-26%, and chlorophyll derivative products of 24-26%. For catalytic pyrolysis, the chemical composition of pyrolysis products comprised protein/amino acid derivative products of 5-15%, carbohydrate derivative products of 18-19.5%, lipid derivative products of 14-27%, and chlorophyll derivative products of 15-20%. The addition of 10% Ni/SBA-15 enhanced the production of aromatic compounds, such as furans, furfurals, alkyl aromatics, and nitrogen aromatic compounds. These were the thermal degradation products of carbohydrates and proteins. However, the amount of fatty acids and phytol fragments in the pyrolysis of Botryococcus braunii decreased in the presence of catalyst. Thermogravimetric analyses showed that the temperature range for the pyrolysis of Botryococcus braunii was 135-547 °C, while that of the catalyzed pyrolysis was 135-532 °C. There was a decrease in pyrolysis yield after incorporating Ni/SBA-15, which may be due to coke formation.

Anti-inflammatory effect of milk whey from different species after in vitro digestion

there is a close relationship between obesity, gut health and immune system. A low-grade of inflammation, which could precede obesity, may have implications for the development of metabolic syndrome and insulin resistance. analyzing the anti-inflammatory capacity of several types of whey (cow, sheep, goat and a mixture of them). an in vitro model of intestinal inflammation employing a cell co-culture (Caco-2 and RAW 264.7) was performed after an in vitro digestion and fermentation (simulating mouth-to-colon conditions). Inflammatory markers such as IL-8 and TNF-α, as well as the transepithelial electrical resistance (TEER) of Caco-2 monolayer, were determined. digested and fermented whey had a protective effect on cell permeability, being lower in the case of fermented goat whey and mixture. The anti-inflammatory activity of whey was greater the more digestion progressed. Fermented whey showed the greatest anti-inflammatory effect, inhibiting IL-8 and TNF-α secretion, probably due to its composition (protein degradation products such as peptides and amino acids, and SCFA). However, fermented goat whey did not show this degree of inhibition, perhaps due to its low SCFA concentration. milk whey, especially after being fermented in the colon, can be useful nutritional strategy to preserve the intestinal barrier and mitigate the low-grade of inflammation that characterizes metabolic disorders and obesity.

Combining mass spectrometry and machine learning to discover bioactive peptides

Peptides play important roles in regulating biological processes and form the basis of a multiplicity of therapeutic drugs. To date, only about 300 peptides in human have confirmed bioactivity, although tens of thousands have been reported in the literature. The majority of these are inactive degradation products of endogenous proteins and peptides, presenting a needle-in-a-haystack problem of identifying the most promising candidate peptides from large-scale peptidomics experiments to test for bioactivity. To address this challenge, we conducted a comprehensive analysis of the mammalian peptidome across seven tissues in four different mouse strains and used the data to train a machine learning model that predicts hundreds of peptide candidates based on patterns in the mass spectrometry data. We provide in silico validation examples and experimental confirmation of bioactivity for two peptides, demonstrating the utility of this resource for discovering lead peptides for further characterization and therapeutic development.

The protective pattern of chitosan-based active coating on texture stabilization of refrigerated carp fillets from the perspective of proteolysis

Herein, the protective pattern of chitosan coating enriched with green tea extract on texture stabilization of refrigerated grass carp fillets was explored. In general, higher shear force and lower endogenous enzyme activities were observed in coated fillets, with the max. level of cathepsins and calpain decreasing by 30.2 ∼ 39.6 % when compared to the control during storage. Meanwhile, the coating reduced protein composition changes and accumulation of protein degradation products. According to label-free proteomic analysis, the proteome closer to fresh sample was observed in coated fillets than that of the control, as supported by PCA and hierarchical cluster analysis. Meanwhile, 33 differential proteins involved in tissue structure, protein phosphorylation and protein turnover were further screened out, and most DAPs showed up-regulation in coated fillets compared to the control on day 12. Presumably, the coating modulated endogenous enzyme-induced myofibrillar protein degradation and protein phosphorylation level, thereby stabilizing the texture properties of refrigerated fillets.

Effect of lignocellulosic components on the hydrothermal carbonization reaction pathway and product properties of protein

Blending lignocellulosic biomass with sewage sludge can further improve the fuel performance of hydrochar. Investigating the effects of different lignocellulosic components on protein hydrothermal carbonization (HTC) reaction pathways and product properties can provide insights into ways to screen lignocellulosic biomass. In this study, cellulose, hemicellulose, and lignin were selected for co-HTC with protein in different mass ratios (1:2 and 2:1). Results showed that the dehydration and decarboxylation reactions were strong when protein–hemicellulose co-HTC in a mass ratio of 2:1, and the O/C and H/C atomic ratios of the hydrochar decreased to 0.14 and 1.19, respectively. As the blending ratio of lignocellulosic components increased, the interaction gradually reached saturation, and the synergistic effect on the properties of mixture hydrochar decreased. Moreover, the functional group characteristics of the mixture hydrochar tended to resemble the chemical structures of lignocellulosic components. The Maillard and Mannich reactions between lignocellulosic components and proteins during co-HTC resulted in the re-solidification of N-containing compounds in the liquid-phase products. The degradation products of protein and (hemi)cellulose (glucose and furfural) were polymerized into char by the Maillard reaction. However, water-soluble N-containing compounds were immobilized into char through adsorption or reaction with lignin O-containing functional groups.

Effect of traditional and dry package ageing on physicochemical properties and protein digestibility of pork Longissimus thoracis muscle

The aim of the study was to evaluate the effect of traditional (unpacked) and dry package ageing on quality characteristics, proteolysis and protein digestibility of pork loins aged for 28 days. Changes in pH and weight loss as a function of ageing were more pronounced in traditionally aged loin in comparison to that aged in a bag. Both traditional and bag ageing induced the enzymatic protein degradation process. However, higher amounts of protein degradation products were observed for in bag aged samples compared to traditionally aged samples. The effect of ageing on protein digestibility was evaluated using a simulated in vitro oral-gastrointestinal digestion model. A significant (P < 0.05) impact of ageing was observed and the protein digestibility of the samples showed an increasing trend with ageing time. This was reflected in the results of peptide concentration and free amino acids which showed a significantly increasing trend (P < 0.05) with increased ageing time. A higher degree of protein hydrolysis and higher concentration of peptides were observed for most of the in bag aged samples compared to traditionally aged samples. These results indicate the superiority of in bag ageing in terms of protein digestibility.

This Week in The Journal

Eunyoung Bang, Annette S. Fincher, Sophie Nader, David A. Murchison, and William H. Griffith (see pages [1020–1034][1]) The aging process involves breakdown of cellular repair processes, disruption of mitochondrial function, accumulation of oxidative damage and protein degradation products,

2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana.

The role of the RNA degradation product 2′,3′-cyclic adenosine monophosphate (2′,3′-cAMP) is poorly understood. Recent studies have identified 2′,3′-cAMP in plant material and determined its role in stress signaling. The level of 2′,3′-cAMP increases upon wounding, in the dark, and under heat, and 2′,3′-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2′,3′-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2′,3′-cAMP treatment. We demonstrated that 2′,3′-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide–adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2′,3′-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2′,3′-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2′,3′-cAMP treatment influences the movement of processing bodies, confirming the role of 2′,3′-cAMP in the formation and motility of membraneless organelles.

Effect of pepsin on antioxidant and antibacterial activity of protein hydrolysate from salted jellyfish (Lobonema smithii and Rhopilema hispidum) by-products

Protein hydrolysates are products of protein degradation that provide various sizes of peptides and free amino acids. Protein hydrolysate from the different types of enzymes and raw materials provides different bioactivity, such as antioxidant and antibacterial activity. Salted jellyfish by-products have the potential to be a source for protein hydrolysate production because of their low price and having collagen protein. This research aimed to evaluate the antioxidant and antibacterial activity of protein hydrolysates from jellyfish by-products. The dried salted jellyfish by-products from the umbrella and oral arm part of white-type (Lobonema smithii) and sand-type (Rhopilema hispidum) were desalted and enzymatically hydrolyzed by 5% (w/w) pepsin for 24 h at 37°C. Bioactivity assays showed that the hydrolysate of the oral arms part of white-type jellyfish exhibited the highest antioxidant activity (13.27%). While protein hydrolysate of umbrella part of sand-type jellyfish showed the highest antibacterial activity against Vibrio parahaemolyticus up to 13.61%. The results demonstrated that peptic hydrolysate of different types and parts of jellyfish by-products provided different antioxidant or antibacterial activity, thereby increasing the potential uses of jellyfish protein hydrolysate as a functional food.