Degradation Of Storage Proteins Research Articles

Sensoproteomic Characterization of Lactobacillus Johnsonii-Fermented Pea Protein-Based Beverage: A Promising Strategy for Enhancing Umami and Kokumi Sensations while Mitigating Bitterness.

This study investigated the mechanism underlying the flavor improvement observed during fermentation of a pea protein-based beverage using Lactobacillus johnsonii NCC533. A combination of sensomics and sensoproteomics approach revealed that the fermentation process enriched or generated well-known basic taste ingredients, such as amino acids, nucleotides, organic acids, and dipeptides, besides six new taste-active peptide sequences that enhance kokumi and umami notes. The six new umami and kokumi enhancing peptides, with human recognition thresholds ranging from 0.046 to 0.555 mM, are produced through the degradation of Pisum sativum's storage protein. Our findings suggest that compounds derived from fermentation enhance umami and kokumi sensations and reduce bitterness, thus improving the overall flavor perception of pea proteins. In addition, the analysis of intraspecific variations in the proteolytic activity of L. johnsonii and the genome-peptidome correlation analysis performed in this study point at cell-wall-bound proteinases such as PrtP and PrtM as the key genes necessary to initiate the flavor improving proteolytic cascade. This study provides valuable insights into the molecular mechanisms underlying the flavor improvement of pea protein during fermentation and identifies potential future research directions. The results highlight the importance of combining fermentation and senso(proteo)mics techniques in developing tastier and more palatable plant-based protein products.

Involvement of a rice mutation in storage protein biogenesis in endosperm and its genomic location.

A mutation was first found to cause the great generation of glutelin precursors (proglutelins) in rice (Oryza sativa L.) endosperm, and thus referred to as GPGG1. The GPGG1 was involved in synthesis and compartmentation of storage proteins. The PPR-like gene in GPGG1-mapped region was determined as its candidate gene. In the wild type rice, glutelins and prolamins are synthesized on respective subdomains of rough endoplasmic reticulum (ER) and intracellularly compartmentalized into different storage protein bodies. In this study, a storage protein mutant was obtained and characterized by the great generation of proglutelins combining with the lacking of 13 kD prolamins. A dominant genic-mutation, referred to as GPGG1, was clarified to result in the proteinous alteration. Novel saccular composite-ER was shown to act in the synthesis of proglutelins and 14 kD prolamins in the mutant. Additionally, a series of organelles including newly occurring several compartments were shown to function in the transfer, trans-plasmalemmal transport, delivery, deposition and degradation of storage proteins in the mutant. The GPGG1 gene was mapped to a 67.256kb region of chromosome 12, the pentatricopeptide repeat (PPR)-like gene in this region was detected to contain mutational sites.

Genome-wide transcriptome analysis reveals key regulatory networks and genes involved in the determination of seed hardness in vegetable soybean.

Seed hardness is an important quality trait of vegetable soybean. To determine the factors underlying seed hardness, two landraces with contrasting seed hardness, Niumaohuang (low seed hardness) and Pixiansilicao (high seed hardness), were selected from 216 soybean accessions originating from 26 provinces in China. The contents of the main components in vegetable soybean seeds such as water, soluble sugar, starch, protein and oil were measured, and transcriptome analyses performed during five stages of seed developmental. Transcriptome analysis indicates that during the middle and late stages of seed development, a large number of genes involved in the synthesis or degradation of starch, storage protein, and fatty acids were differentially expressed, leading to differences in the accumulation of stored substances during seed maturation among Niumaohuang and Pixiansilicao. The activity of cell proliferation and the formation of cell walls in the middle and late stages of seed development may also affect the hardness of seeds to a certain extent. In addition, weighted gene co-expression network analysis (WGCNA) was undertaken to identify co-expressed gene modules and hub genes that regulate seed hardness. Overexpression of a candidate seed hardness regulatory hub gene, GmSWEET2, resulted in increased seed hardness. In this study, the important role of GmSWEET2 in regulating the hardness of vegetable soybean seeds was verified and numerous potential key regulators controlling seed hardness and the proportion of seed components were identified, laying the groundwork for improving the texture of vegetable soybean.

Integrated proteome and physiological traits reveal interactive mechanisms of new leaf growth and storage protein degradation with mature leaves of evergreen citrus trees.

The growth of fruit trees depends on the nitrogen (N) remobilization in mature tissues and N acquisition from the soil. However, in evergreen mature citrus (Citrus reticulata Blanco) leaves, proteins with N storage functions and hub molecules involved in driving N remobilization remain largely unknown. Here, we combined proteome and physiological analyses to characterize the spatiotemporal mechanisms of growth of new leaves and storage protein degradation in mature leaves of citrus trees exposed to low-N and high-N fertilization in the field. Results show that the growth of new leaves is driven by remobilization of stored reserves, rather than N uptake by the roots. In this context, proline and arginine in mature leaves acted as N sources supporting the growth of new leaves in spring. Time-series analyses with gel electrophoresis and proteome analysis indicated that the mature autumn shoot leaves are probably the sites of storage protein synthesis, while the aspartic endopeptidase protein is related to the degradation of storage proteins in mature citrus leaves. Furthermore, bioinformatic analysis based on protein-protein interactions indicated that glutamate synthetase and ATP-citrate synthetase are hub proteins in N remobilization from mature citrus leaves. These results provide strong physiological data for seasonal optimization of N fertilizer application in citrus orchards.

The effects of germination on the composition and functional properties of hemp seed protein isolate

The effects of germination on the functional properties of hemp seed protein isolates was evaluated in this study. Significant seed storage protein degradation was observed after three days of germination, while protein surface charge was altered after one day of germination. Protein solubility increased in neutral to slightly acidic pH, with 5-day germination increasing hemp seed protein solubility from 10.3% to 21.8% at pH 7.0. Water holding capacity increased from 1.03 g H 2 O/g isolate to 1.34 g H 2 O/g isolate while oil holding capacity stayed relatively constant. Germinated protein isolates demonstrated higher foaming capacity (69.2%–111.2%) and foaming stability (53.6%–75.1%) than non-germinated protein isolates. Five-day germinated hemp seed protein isolate was the only adequate emulsifier in this study, capable of stabilizing emulsion droplets down to 2.90 μm in diameter. Hemp seed protein formed self-supporting gels at minimum of 2 or 3 w/v %, and germination increased the hardness of hemp seed protein gels (38.02 g–49.12 g for 10 w/v % gels). These findings indicate the ability to leverage germination as a clean-label process for improving the functional properties of hemp seed protein. • Hemp seed storage proteins are degraded after three days of germination. • Germinated hemp seed protein isolates are more soluble near a neutral pH. • Germination improves hemp protein WHC, foaming, emulsifying, and gelling properties. • LGC for hemp protein at pH 7 was determined at 2–3 w/v %.

Effect of Solvent Type and Germination Time on The Level of Free Amino Acid and Peptides of Germinated Pigeon Pea (Cajanus cajan (L.) Millsp) Extract

Germination can activate the degradation of storage protein in legumes releasing peptides and free amino acids for seed growth. These compounds have many benefits in many fields, especially in food and health. This study aimed to determine the effect of solvent type and germination time on the level of free amino acids (FAA) and the level of peptides (MW < 10 kDa) of germinated pigeon pea extract. The extraction of free amino acids and the dissolved protein from germinated pigeon pea flour was carried out using two kinds of solvents, namely water, and 0.1 N HCl. The variation of germination times of pigeon pea seeds was carried out at 12, 24, 36, 48, 60, 72, 84, and 96 hours. The level of FAA was determined spectrophotometrically using the ninhydrin method after the deproteination step, while the level of peptides was determined spectrophotometrically using the biuret method. The results showed that using 0.1 N HCl as a solvent produced a higher level of FAA and peptides in the extract than using water. The increase of germination time can increase the level of FAA with the peak achieved in 36 hours. The increase of germination time can increase the level of peptides with the peak of peptides level achieved on 84 hours of germination. These results showed that seed germination is a potential method for producing free amino acids and peptides which accumulate at a specific time

StresSeed: The Unfolded Protein Response During Seed Development.

During seed development, the endoplasmic reticulum (ER) takes care of the synthesis and structural maturation of very high amounts of storage proteins in a relatively short time. The ER must thus adjust its extension and machinery to optimize this process. The major signaling mechanism to maintain ER homeostasis is the unfolded protein response (UPR). Both storage proteins that assemble into ER-connected protein bodies and those that are delivered to protein storage vacuoles stimulate the UPR, but its extent and features are specific for the different storage protein classes and even for individual members of each class. Furthermore, evidence exists for anticipatory UPR directly connected to the development of storage seed cells and for selective degradation of certain storage proteins soon after their synthesis, whose signaling details are however still largely unknown. All these events are discussed, also in the light of known features of mammalian UPR.

Integrated physiological, proteome and gene expression analyses provide new insights into nitrogen remobilization in citrus trees

Nitrogen (N) remobilization is an important physiological process that supports the growth and development of trees. However, in evergreen broad-leaved tree species, such as citrus, the mechanisms of N remobilization are not completely understood. Therefore, we quantified the potential of N remobilization from senescing leaves of spring shoots to mature leaves of autumn shoots of citrus trees under different soil N availabilities and further explored the underlying N metabolism characteristics by physiological, proteome and gene expression analyses. Citrus exposed to low N had an approximately 38% N remobilization efficiency (NRE), whereas citrus exposed to high N had an NRE efficiency of only 4.8%. Integrated physiological, proteomic and gene expression analyses showed that photosynthesis, N and carbohydrate metabolism interact with N remobilization. The improvement of N metabolism and photosynthesis, the accumulation of proline and arginine, and delayed degradation of storage protein in senescing leaves are the result of sufficient N supply and low N remobilization. Proteome further showed that energy generation proteins and glutamate synthase were hub proteins affecting N remobilization. In addition, N requirement of mature leaves is likely met by soil supply at high N nutrition, thereby resulting in low N remobilization. These results provide insight into N remobilization mechanisms of citrus that are of significance for N fertilizer management in orchards.

Aquaporin activity of barley tonoplast intrinsic proteins is involved in the delay of the coalescence of protein storage vacuoles in aleurone cells

The coalescence of protein storage vacuoles (PSVs) is one of the most prominent cellular changes occurring in cereal aleurone cells during germination. This structural change is highly coupled with the functional transition of this organelle from a storage compartment to a lytic section. Gibberellic acid (GA) promotes this process, whereas abscisic acid (ABA) prevents it. Previously, we demonstrated that ABA-inducible HvTIP3;1 plays a decisive role in ABA-mediated prevention of PSV fusion. In this follow-up study, we examined whether the aquaporin activity of tonoplast intrinsic protein (TIP) is related to its preventive effect on PSV fusion using various functional mutants. The defective forms of aquaporin (HvTIP3;1m and HvTIP3;1ΔNPA-GFPs for HvTIP3;1, and HvTIP1;2m for HvTIP1;2) were found to be less effective than the usual form in delaying the PSV fusion process occurring in GA-treated cells. In contrast, overexpression of HvTIP3;1m reduced the preventive effect of ABA on PSV fusion. Upon inhibition of aquaporin activity using mercury, PSV fusion occurred to a greater extent in ABA-treated barley protoplasts. These data suggest that the aquaporin activity of TIP is involved in the deterrent effect of TIP on PSV coalescence. TIP3-GFP barley transgenic seeds showed prolonged expression of the TIP3;1 transcript. Moreover, PSV fusion progressed at a much slower rate compared to wild type. Additionally, the degradation of storage proteins was not as efficient, suggesting that a metamorphic transition of PSVs to lytic organelles is closely correlated with the disappearance of HvTIPs and the PSV fusion process.

Loss of viability and vigour in the course of short-term storage of Pityrocarpa moniliformis seeds cannot be attributed to reserve degradation

The quality of forest seeds may be maintained in the course of storage for their use in the restoration of degraded areas and the conservation of genetic resources. In this sense, physiological and biochemical markers were evaluated during the short-term storage of Pityrocarpa moniliformis seeds, a pioneer species native to the Caatinga. Seeds harvested from 25 mother trees were stored in a growth chamber (27 ± 4 °C) or in a refrigerator (6 ± 3 °C) for 360 days. Viability, vigour, reserve contents, metabolite contents, and hydrolase activities were assessed at the beginning of the experiment and every 60 days. Over time, a decrease in germination percentage, germination speed index, and seedling growth accompanied by an increase in membrane damage was verified in the seeds kept in a growth chamber. These alterations were not associated with the degradation of starch and storage proteins, although there was a decrease in the content of non-reducing sugars in the seeds kept in the growth chamber. Therefore, the loss of viability and vigour in P. moniliformis seeds stored for 360 days in a growth chamber cannot be attributed to the hydrolysis of major reserves, but it may be related to the accumulation of reducing sugars.

Natural Saponin Formosanin C‐induced Ferroptosis in Human Hepatocellular Carcinoma Cells Involved Ferritinophagy

Formosanin C (FC) is an anti‐tumor diosgenin saponin isolated from Paris formosana Hayata (Liliaceae) which is a perennial herb grown in the humid and dark forests of Taiwan at about the level of 1000 m. The plat has been used as a folk remedy for snake‐bite inflammation and tumors. FC is also a component of “Yunnan Bai Yao”, a traditional Chinese hemostatic medicine. Ferroptosis is a new form of iron‐dependent cell death, which occurs via an increased labile iron pool and lipid reactive oxygen species (ROS). Ferritinophagy is a ferritin (an iron storage protein) degradation pathway, depending on a selective autophagic cargo receptor nuclear coactivator 4 (NCOA4). Increased labile iron pool by ferritinophagy has been recognized as an upstream regulator of ferroptosis. Thus, ferroptosis has been reported to be a form of autophagic cell death. Although iron is an essential nutrient that facilitates cell proliferation and growth, overexpression of ferritin and down‐regulation of ferroportin (an iron export protein) has recently been observed in human hepatocellular carcinoma (HCC), indicating the increased dependence of iron in the cancer cells. In the present study, FC caused a significant dosage‐related population growth inhibition in HCC HepG2 and Hep3B cells using SRB assay, which can be partially blocked by co‐administration of ferroptosis inhibitor ferrostatin‐1 (a scavenger of lipid ROS). FC induced elevation in lipid ROS formation can also be partially reversed by addition of either ferrostatin‐1, or the presence of antioxidant vitamin C and/or vitamin E using flow cytometry, confirming FC‐induced ferroptosis. Flow cytometric analysis also revealed that FC elevated the percentage of acidic vesicular organelles‐positive cells, representing the formation of autolysosomes and induction of autophagic flux in both cell lines. The FC‐induced autophagy was confirmed using Western blotting, characterized by the production of LC3‐II and a further elevation of LC3‐II expression by addition of autophagy inhibitor bafilomycin A1 to prevent the fusion of autophagosomes and lysosomes for cargo degradation. In the present of bafilomycin A1, accumulation of NCOA4 and ferritin was observed in both FC‐treated cell lines using Western blotting. Concurrently, increase in co‐localization of LC3 puncta with NCOA4 or ferritin was exhibited using confocal microscopy, suggesting FC‐induced ferritinophagy. It is noteworthy that HepG2 cells with a higher NCOA4 expression were more sensitive to FC‐induced ferroptosis and autophagy compared to Hep3B cells with a higher ferritin expression. Overall, our data suggest chemotherapeutic potential of phytochemical FC for treatment of HCC with higher expression of NCOA4 via ferritinophagy and ferroptosis.Support or Funding InformationSupported in part by the Ministry of Science and Technology, Taiwan, No. MOST 106‐2320‐B‐003‐006‐MY3 and 107‐2320‐B‐003‐002‐MY2

Angiotensin Converting Enzyme (ACE) Inhibitory Activity of Peptide Fraction of Germinated Pigeon Pea (<i>Cajanus cajan</i> (L.) Millsp.)

During the germination process, seeds can release various types of peptides due to the degradation of storage proteins. Some of these peptides can have biological activity (bioactive peptides). The objective of this study was to determine the ACE inhibitory activity of germinated pigeon pea peptide extract at various germination times and to carry out the fractionation to the extract to get the most active peptide fraction. The results showed that the highest activity of peptide extract was found on the 4th-day germination of pigeon pea with an IC50 value of 63.46 μg/mL. The peptide extract was further fractionated by centrifugal ultrafiltration method and it was found that the peptide fraction < 3 kDa had the highest ACE inhibitory activity with an IC50 value of 57.79 μg/mL. The result of identification with the LCMS method to the fraction was able to detect 4 types of the peptide with a molecular weight of 230.304, 294.303, 441.436, and 570.591 Da. These results suggested that the peptide fraction of germinated pigeon pea has the potency as an ACE inhibitory nutraceutical.

UHPLC–MS/MS analysis of cocoa bean proteomes from four different genotypes

In this study the proteomic profiles of cocoa beans from four genotypes with different flavour profiles were analysed by bottom-up label-free UHPLC–MS/MS. From a total of 430 identified proteins, 61 proteins were found significantly differentially expressed among the four cocoa genotypes analysed with a fold change of ≥2. PCA analysis allowed clear separation of the genotypes based on their proteomic profiles. Genotype-specific abundances were recorded for proteases involved in the degradation of storage proteins and release of flavour precursors. Different genotype-specific levels of other enzymes, which generate volatiles compounds that could potentially lead to flavour-inducing compounds, were also detected. Overall, this study shows that UHPLC-MS/MS data can differentiate cocoa bean varieties.

Identification of a type II cystatin in Fragaria chiloensis: A proteinase inhibitor differentially regulated during achene development and in response to biotic stress-related stimuli

The equilibrium between protein synthesis and degradation is key to maintaining efficiency in different physiological processes. The proteinase inhibitor cystatin regulates protease activities in different developmental and physiological contexts. Here we describe for the first time the identification and the biological function of the cysteine protease inhibitor cystatin of Fragaria chiloensis, FchCYS1. Based on primary sequence and 3D-structural homology modelling, FchCYS1 is a type II phytocystatin with high identity to other cystatins of the Fragaria genus. Both the papain-like and the legumain-like protease inhibitory domains are indeed functional, based on in vitro assays performed with Escherichia coli protein extracts containing recombinant FchCYS1. FchCYS1 is differentially-expressed in achenes of F. chiloensis fruits, with highest expression as the fruit reaches the ripened stage, suggesting a role in preventing degradation of storage proteins that will nourish the embryo during seed germination. Furthermore, FchCYS1 responds transcriptionally to the application of salicylic acid and to mechanical injury, strongly suggesting that FchCYS1 could be involved in the response against pathogen attack. Overall these results point to a role for FchCYS1 in diverse physiological processes in F. chiloensis.

The deterioration of Moringa oleifera Lam. seeds in the course of storage involves reserve degradation

Seed deterioration in the course of storage may involve hydrolytic reactions. Hence, we aimed to evaluate viability, vigour, contents of reserves and metabolites, and activities of hydrolytic enzymes in Moringa oleifera Lam. seeds during storage under controlled conditions. Seeds were packaged in semipermeable plastic and maintained in a growth chamber (27 ± 2 °C and RH 60–65%) and under refrigeration (4 ± 2 °C and RH 20–25%) for 18 months. Samples were taken at the start of the experiment and every 3 months. During the first 12 months, water content, viability, and vigour remained almost unaffected, while the content of neutral lipids, starch, soluble sugars and free amino acids did not reduce in the seeds kept under refrigeration. After this period, the loss of viability and vigour was accompanied by the degradation of storage lipids, storage proteins, and non-reducing sugars associated with the increase of lipase and acid protease activity in both environmental conditions. As the seed water content remained below 8% in the course of the experiment, we suggest that non-enzymatic hydrolysis might play a role in the deterioration of M. oleifera seeds during storage. At least for planting, we recommend that M. oleifera seeds be kept at low relative humidity under refrigeration for up to 12 months.

Proteomic analysis reveals a role of melatonin in promoting cucumber seed germination under high salinity by regulating energy production

Seed germination is a critical and complex process in the plant life cycle. Although previous studies have found that melatonin can promote seed germination under salt stress, the involvement of melatonin in the regulation of proteomic changes remains poorly understood. In this study, a total of 157 proteins were significantly influenced (ratio ≥ 2 or ≤ −2) by melatonin during seed germination under salt stress using a label-free quantitative technique. Our GO analysis revealed that several pathways were obviously regulated by melatonin, including ribosome biosynthesis, lipid metabolism, carbohydrate metabolism, and storage protein degradation. Not only stress-tolerant proteins but also proteins that produce ATP as part of glycolysis, the citric acid cycle, and the glyoxylate cycle were upregulated by melatonin. Overall, this study provides new evidence that melatonin alleviates the inhibitory effects of NaCl stress on seed germination by promoting energy production. This study is the first to provide insights at the proteomic level into the molecular mechanism of melatonin in response to salt stress in cucumber seeds. This may be helpful to further understand the role of melatonin in cucumber seed germination under stress conditions.

Proteome analysis reveals an energy-dependent central process for Populus × canadensis seed germination

Poplar (Populus×canadensis) seeds rapidly germinated in darkness at 10, 15, and 20°C and reached 50% seed germination after about 22, 4.5, and 3.5h, respectively. Germination of poplar seeds was markedly inhibited by abscisic acid (ABA) at 50μM and cycloheximide (CHX) at 100μM, and these inhibitive roles were temperature-dependent. In the present study, mature poplar seeds were used to investigate the differentially changed proteome of seeds germinating in water, ABA, and CHX. A total of 130 protein spots showed a significant change (1.5-fold increase/decrease, P<0.05) in abundance, and 101 protein spots were successfully identified. Most of the proteins were associated with cell defense and rescue (21%), storage proteins (21%), protein synthesis and destination (20%), metabolism (16%), and energy (14%). The germination of poplar seeds is closely related with the increase in those proteins involved in amino acid and lipid metabolism, the tricarboxylic acid cycle and pentose phosphate pathway, protein synthesis and destination, cell defense and rescue, and degradation of storage proteins. ABA and CHX inhibit the germination of poplar seeds by decreasing the protein abundance associated with protein proteolysis, protein folding, and storage proteins. We conclude that poplar seed germination is an energy-dependent active process, and is accompanied by increasing amino acid activation, protein synthesis and destination, as well as cell defense and rescue, and degradation of storage proteins.

Aktivita proteolytických enzymů v průběhu sladování a výroby piva

Malting is controlled germination of barley grain under certain conditions to the time of grain modifi cation and it is a key process of the brewing and distilling industries. The aim of malting is to produce from barley malt containing the necessary enzymes, aroma and color substances needed for the production of a certain type of beer. An important part of this process is the activity of proteolytic enzymes, not only during the degradation of storage proteins but also in other aspects of the germination process, for example activation of other enzymes. Grain modifi cation is an important criterion for the quality of produced malts and subsequently the quality and specifi c characteristics of different types of beers.

Proteome changes associated with dormancy release of Dongxiang wild rice seeds

Seed dormancy provides optimum timing for seed germination and subsequent seedling growth, but the mechanism of seed dormancy is still poorly understood. Here, we used Dongxiang wild rice (DXWR) seeds to investigate the dormancy behavior and the differentially changed proteome in embryo and endosperm during dormancy release. DXWR seed dormancy was caused by interaction of embryo and its surrounding structure, and was an intermediate physiological dormancy. During seed dormancy release, a total of 109 and 97 protein spots showed significant change in abundance and were successfully identified in embryo and endosperm, respectively. As a result of dormancy release, the abundance of nine proteins involved in storage protein, cell defense and rescue and energy changed in the same way in both embryo and endosperm, while 67 and 49 protein spots changed differentially in embryo and endosperm, respectively. Dormancy release of DXWR seeds was closely associated with degradation of storage proteins in both embryo and endosperm. At the same time, the abundance of proteins involved in metabolism, glycolysis and TCA cycle, cell growth and division, protein synthesis and destination and signal transduction increased in embryos while staying constant or decreasing in endosperms.

Insulin-induced changes in metabolism-related proteins during maize germination

Insulin regulates a wide range of metabolic processes in mammals, such as homeostasis and the breakdown of glucose. Recently, the existence of an insulin-related growth factor in maize (ZmIGF) and a possible receptor for this growth factor has been reported. This peptide exerts effects on plant growth and promotes germination by activating the target of rapamycin (TOR) signaling pathways, which is similar to the insulin response in mammals. In this study, we analyzed the insulin response in maize embryos using a proteomic approach. Our results indicated that insulin modulates the expression of proteins involved in processes, such as storage protein degradation, protein processing, redox and desiccation stress, and glucose metabolism. The involvement of TOR signaling pathways was analyzed using the TOR inhibitor, rapamycin. The results showed that the modulation of these proteins by insulin is independent of the TOR pathway. These results indicated that insulin promotes changes in metabolism-related proteins to ensure successful germination in maize.