Lysine-rich Proteins Research Articles

Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins

Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine’s ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine’s ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems.

A review of nutritional profile and processing of faba bean (Vicia faba L.)

AbstractFaba bean (Vicia faba L.), a nutritious leguminous cool tolerant crop, is widely cultivated throughout the world. China, Ethiopia, the United Kingdom, Australia, and France are the main producers of faba beans. In recent years, interest has been growing in health and nutritional benefits of faba beans and developments of different foods enriched with biomolecules with improved functionality, nutrition value, and health benefits. Faba beans are rich source of lysine rich proteins, carbohydrate, minerals, vitamins, and numerous bioactive compounds. It is also a good source of l‐3,4‐dihydroxyphenylalanine (L‐DOPA), which is a precursor of dopamine and can be potentially utilized for Parkinson's disease treatment. The seeds of faba beans can be consumed dry, roasted, soaked, cooked, frozen, or canned. However, a number of antinutritional factors such as phytic acid, trypsin inhibitors, saponins, vicin and convicine (favism‐inducing compounds), lectins, and condensed tannins negatively affect the biological value of faba beans resulting in its underutilization. For expanding the utilization of faba beans in human nutrition, the removal of these antinutrients is necessary. A number of methods including dehulling, soaking, germination, fermentation, and heat processing (cooking, boiling, extrusion, and autocalving) have been used individually or in combination to eliminate or destroy the antinutritional factors in faba beans. This comprehensive review covers global production, nutritional profile, and processing of faba beans and its utilization in various product developments.

Role of the lysyl oxidase enzyme family in cardiac function and disease

Heart diseases are a major cause of morbidity and mortality world-wide. Lysyl oxidase (LOX) and related LOX-like (LOXL) isoforms play a vital role in remodelling the extracellular matrix (ECM). The LOX family controls ECM formation by cross-linking collagen and elastin chains. LOX/LOXL proteins are copper-dependent amine oxidases that catalyse the oxidation of lysine, causing cross-linking between the lysine moieties of lysine-rich proteins. Dynamic changes in LOX and LOXL protein-expression occur in a variety of cardiac pathologies; these changes are believed to be central to the associated tissue-fibrosis. An awareness of the potential pathophysiological importance of LOX has led to the evaluation of interventions that target LOX/LOXL proteins for heart-disease therapy. The purposes of this review article are: (i) to summarize the basic biochemistry and enzyme function of LOX and LOXL proteins; (ii) to consider their tissue and species distribution; and (iii) to review the results of experimental studies of the roles of LOX and LOXL proteins in heart disease, addressing involvement in the mechanisms, pathophysiology and therapeutic responses based on observations in patient samples and relevant animal models. Therapeutic targeting of LOX family enzymes has shown promising results in animal models, but small-molecule approaches have been limited by non-specificity and off-target effects. Biological approaches show potential promise but are in their infancy. While there is strong evidence for LOX-family protein participation in heart failure, myocardial infarction, cardiac hypertrophy, dilated cardiomyopathy, atrial fibrillation and hypertension, as well as potential interest as therapeutic targets, the precise involvement of LOX-family proteins in heart disease requires further investigation.

Mutations in KARS cause a severe neurological and neurosensory disease with optic neuropathy.

Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.

L-Lysine as the Molecule Influencing Selective Brain Activity in Pain-Induced Behavior of Rats.

Lysine-rich proteins are some of the most important proteins of neurons and it has become necessary to investigate the possible role of L-lysine as a brain functioning regulator. The purpose of our study is to identify the characteristics and the mechanisms of L-lysine effects on the different types of pain-induced behavior in the stimulation of tail and foot-shock models in 210 adult male Wistar rats. L-lysine was administered in intraperitoneal or intracerebroventricular injections in doses of 0.15–50.0 µg/kg. When a tail is irritated, L-lysine was found to enhance pain sensitivity and affective defense after both intraperitoneal and intracerebroventricular administration. In the case of unavoidable painful irritation of a pair of rats with both types of L-lysine administration, there was no direct correlation of the severity of pain with defensive reactions and outbursts of aggression. This indicates a more complex integration of the activity of brain structures in this situation of animal interaction, which was confirmed by the results of the direct amino acid action on the periventricular brain structures. Our findings show that L-lysine influences the selective brain activity in dependence on the biological significance of pain-induced behavior.

Gene expression of an arabinogalactan lysine-rich protein CaAGP18 during vegetative and reproductive development of bell pepper (Capsicum annuum L.).

Lysine-rich (Lys-rich) proteins encoded by AGP17, AGP18, and AGP19 genes are cell wall-associated glycopeptides related to sexual reproduction in flowering plants. This subclass belongs to classical arabinogalactan proteins (AGPs) widely studied in model plants like Arabidopsis. In this study, we identified the CaAGP18 cDNA from bell pepper (Capsicum annuum L.), as well as its expression pattern during vegetative and reproductive development. The deduced amino acid sequence revealed a Lys-rich AGP18 protein of 238 amino acids residues in length with an estimated molecular mass of 22.85kDa and an isoelectric point of 9.7. The protein is predicted as canonical AGP due to the presence of a small Lys-rich region and a C-terminal sequence essential for posttranslational modification with a glycosylphosphatidylinositol (GPI). Phylogenetic analysis showed that CaAGP18 is clustered together with NtAGP18, SpAGP18, StAGP18 and NaAGP18 from Solanaceae species. CaAGP18 expression through plant phenological stages had the highest transcription level in leaves at the seedling stage, whereas in reproductive organs there was a significant up-regulation in pistils during anthesis, also in petals 2days post-anthesis (DPA), and in fruit at the expansion stage. Our results open future research for possible roles of CaAGP18 in cell expansion as a wall-associated plasticizer and reproductive processes like pistil interactions and petal cell death.

Girardia dorotocephala transcriptome sequence, assembly, and validation through characterization of piwi homologs and stem cell progeny markers

Planarian flatworms are popular models for the study of regeneration and stem cell biology in vivo. Technical advances and increased availability of genetic information have fueled the discovery of molecules responsible for stem cell pluripotency and regeneration in flatworms. Unfortunately, most of the planarian research performed worldwide utilizes species that are not natural habitants of North America, which limits their availability to newcomer laboratories and impedes their distribution for educational activities. In order to circumvent these limitations and increase the genetic information available for comparative studies, we sequenced the transcriptome of Girardia dorotocephala, a planarian species pandemic and commercially available in North America. A total of 254,802,670 paired sequence reads were obtained from RNA extracted from intact individuals, regenerating fragments, as well as freshly excised auricles of a clonal line of G. dorotocephala (MA-C2), and used for de novo assembly of its transcriptome. The resulting transcriptome draft was validated through functional analysis of genetic markers of stem cells and their progeny in G. dorotocephala. Akin to orthologs in other planarian species, G. dorotocephala Piwi1 (GdPiwi1) was found to be a robust marker of the planarian stem cell population and GdPiwi2 an essential component for stem cell-driven regeneration. Identification of G. dorotocephala homologs of the early stem cell descendent marker PROG-1 revealed a family of lysine-rich proteins expressed during epithelial cell differentiation. Sequences from the MA-C2 transcriptome were found to be 98–99% identical to nucleotide sequences from G. dorotocephala populations with different chromosomal number, demonstrating strong conservation regardless of karyotype evolution. Altogether, this work establishes G. dorotocephala as a viable and accessible option for analysis of gene function in North America.

Technological, nutritional and functional properties of wheat bread enriched with lentil or carob flours

Blending wheat flour with carob seed or green lentil flour at 5–6%, 10–12% or 24% level modified the dough's technological properties and bread characteristics. Carob seed flours increased dough tenacity while reducing extensibility, whereas lentil flour reduced dough tenacity, extensibility and strength. Carob seed flours increased water absorption up to about 40%. Flours from all legumes increased dough development time (carob flours more than lentils flour). Lentil flour strongly decreased the stability due to weakening of the gluten network. In a baking test, however, all blends gave acceptable loaves. Blending the wheat flour with 5–6% legume flour generally did not alter the loaf volume. However, increasing the legume flour to 10–12% or 24% reduced the loaf volume, except when supplemented with refined carob seed flour at 10%, which increased it. Carob flour and especially lentil flour enriched bread with lysine-rich proteins, dietary fibre, phenolic compounds and lignans and in general increased its antioxidant power.The nutritional value of lentils and the technological properties of carob are useful in increasing nutritional and functional value of wheat bread.

Cloning a lysine-rich protein gene from potato (Solanum tuberosum L.) cultivar Thuong Tin and construction of the expression vector

Lysine is one of the limiting essential amino acids because it is not synthesized in the body of animals and humans. They must obtain lysine from their diet. Recent results of gene transfer research showed the possibility of overexpression of genes encoding natural lysine-rich proteins in crops such as rice and corn, to improve protein quality by increasing the lysine content. However, there has been little report on cloning genes for lysine-rich proteins. In this article, we present the results of cloning the StTTLP gene encoding a lysine-rich protein from Thuong Tin potato cultivar. After successful cloning, we have constructed an expression vector to be used for gene transfer. The cloned gene had similarities of 94% and 99% to the SBgLR sequences that were registered in GenBank with the accession numbers KU987257.1 and AY377987.1, respectively. The deduced amino acid sequence of StTTLP protein has high lysine proportion of 16.9%. In addition, glutamic acid component was also high with the value of 22.8%. Thus, the cloned gene is considered as the gene encoding a lysine- and glutamic acid-rich protein. StTTLP gene was successfully cloned into the plant expression vector pCAMBIA2300 under the control of globulin 1 promoter (Glo1) from maize. These results provide a useful tool for gene engineering to improve the quality of protein in crop plants.

Proteomic analysis of mature barley grains from C-hordein antisense lines

Hordeins are the major storage proteins in barley grains and are responsible for their low nutritional quality. Previously, antisense C-hordein barley lines were generated and were shown to contain a more balanced amino acid composition and an altered storage protein profile. In the present study, a proteomic approach that combined two-dimensional gel electrophoresis (2-DE) and mass spectrometry was used to (1) identify the changes in the protein profile of non-storage proteins (salt soluble fraction) in antisense C-hordein barley lines (L1, L2 and L3) and (2) map the differentially expressed proteins compared to the non-transgenic control line (Hordeum vulgare cv. Golden Promise). Moreover, the changes in the proteins were correlated with the more balanced amino acid composition of these lines, with special attention to the lysine content. The results showed that suppression of C-hordein expression does not exclusively affect hordein synthesis and accumulation. The more balanced amino acid composition observed in the transgenic lines L1, L2 and L3 was an indirect result of the profound alterations in the patterns of the non-storage proteins. The observed changes included up-regulated expression of the proteins involved in stress and detoxification (L1), defence (L2 and L3), and storage globulins (L1, L2 and L3). To a lesser extent, the proteins involved in grain metabolism were also changed. Thus, the increased essential amino acids content results from changes in distinct protein sources among the three antisense C-hordein lines analyzed, although the up-regulated expression of lysine-rich proteins was consistently observed in all lines.

Investigation of Novel Spectroscopic Features in the Near Ultraviolet Region Arising from Non-Aromatic Amino Acids in Peptides and Proteins

Absorption in the UV region has been dominated by aromatic amino acids in peptides and proteins due to the presence of a conjugated structure in their side chains. However, L-Lysine monohydrochloride displayed new near ultraviolet (270 nm) absorption spectra at high concentrations (0.5 M) in aqueous medium [1]. Similar spectral signatures have been reported for lysine rich proteins such as Human Serum Albumin [2]. The origin of these novel and intriguing spectral features in proteins can only be revealed in the absence of any aromatic moiety.We present here the experimental investigations on absorption spectra of amine containing non-aromatic compounds, lysine containing short peptides in aqueous medium and a lysine rich protein (Alpha3C) which lacks aromatic amino acids in order to elucidate the role of the NH2 moiety behind these spectral properties.We carried out excited state TDDFT electronic structure calculations on lysine residues sampled from classical atomistic molecular dynamics simulations of Alpha3C (PDB Code: 2LXY). The calculated UV-VIS spectra of lysine residues from Alpha3C indicate absorption features above 250 nm in agreement with experiments. The unique spectral signatures of lysine residues are shown to arise from charge transfer and pi-pi∗ transitions involving both, the NH3(+) head group and the peptide backbone. The long tail of the experimental absorption spectra above 250 nm is attributed to the sensitivity of the charge transfer and pi-pi∗ transitions to the lysine conformation and environment. Taken together, our joint experimental and theoretical investigation adds a new dimension towards the understanding of UV-VIS absorption in proteins and amino acids.

Abstract 310: Oxidized LDL-binding Cationic Peptides and Proteins in Inflammation and Atherosclerosis: Novel Insights

Background: Apolipoprotein A1 and apolipoprotein E mimetic peptides have attracted attention due to their ability to reduce atherosclerosis and exhibit antioxidant, anti-inflammatory, and hypolipidemic properties. We previously reported that the non-lipoprotein-related Lys-Arg-rich cationic peptides have anti-inflammatory properties both in vitro and in vivo. In this study, we have determined whether ApoB100 of low density lipoprotein (N-LDL), which is Lys-Arg rich, has similar properties. Methods: 5F-mimetic peptide of ApoA1, LL27 derived from the anti-microbial peptide CAMP, and a human glycodelin derived peptide, were commercially synthesized. N-LDL was prepared and used. The ability of these peptides and protein to neutralize charges of modified lipoproteins, as well as attenuate macrophage uptake and inflammation, were analyzed. Oxidized LDL (Ox-LDL) was pretreated with increasing concentrations of peptides and N-LDL to evaluate charge neutralizing properties of the peptides as well as that of the protein (ApoB100). RAW cells were incubated with LPS or Ox-LDL pretreated with and without peptides and N-LDL. RNA was isolated from treated cells and real time PCR was performed using mouse IL-1α and IL-6 primers. Results: Cationic peptides as well as ApoB100 protein of N-LDL decomposed the peroxide content of 13-HPODE. Incubation of Ox-LDL and Ac-LDL with the peptides as well as ApoB100 resulted in charge neutralization as noted by agarose gel electrophoresis. Pre-incubation of the peptides and N-LDL with modified lipoproteins reduced the uptake of the latter by macrophages and foam cell formation as detected by Oil-Red O staining. Reduced inflammation was observed in the presence of N-LDL as compared to LPS/Ox-LDL. Conclusions: Based on these studies, we postulate that cationic peptides and protein might have properties that would a) affect events that are unrelated to lipid lowering, b) might play an additional role in immune competent cells, including macrophages, and c) might interact with other biologically important anionic molecules, including lipids and proteins. We also predict that lysine-rich cationic peptides and proteins could have therapeutic potential in reducing CVD/atherosclerosis-associated inflammation.

Role of early glycation Amadori products of lysine-rich proteins in the production of autoantibodies in diabetes type 2 patients.

In diabetes, protein glycation mostly occurs at intrachain lysine residues resulting in the formation of early stage Amadori products which are finally converted to advance glycation end products (AGEs). Several studies have reported autoantibodies against AGEs in diabetes but not much data are found in respect of Amadori products. In this study, poly-L-lysine (PLL) was glycated with 50 mM glucose and the resultant Amadori products were estimated by fructosamine or nitroblue tetrazolium assay. We report high content of Amadori products in PLL upon glycation. Glycated PLL showed marked hyperchromicity in the UV spectrum, ellipticity changes in CD spectroscopy, and variations in ε-methylene protons shift in NMR. It was better recognized by autoantibodies in type 2 diabetics compared to the native PLL. Induced antibodies against glycated PLL were successfully used to probe early glycation in the IgG isolated from diabetes type 2 patients. Role of Amadori products of glycated proteins in the induction of autoantibodies in type 2 diabetes as well as in associated secondary complications has been discussed.

Sulfo‐NHS‐SS‐biotin derivatization: A versatile tool for MALDI mass analysis of PTMs in lysine‐rich proteins

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.

A New Method for Quantitative Immunoblotting of Endogenous α-Synuclein

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.

Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum

Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.

Identification and characterization of lysine-rich proteins and starch biosynthesis genes in the opaque2mutant by transcriptional and proteomic analysis

BackgroundThe opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary.ResultsIn this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch.ConclusionsThe results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm.

Preliminary studies of a lysine-rich protein gene transferred into rice

The nutritional quality of proteins in cereals such as rice,maize, wheat, and other cropsis comparatively low because of a deficiency in some essential amino acids. Lysine is the first restrictive amino acid in these cereals. Lysine-rich proteins were isolated and purified from seeds of the winged bean ( Psophocarpustetragonolobus ), alegume. According to N- and C amino acid sequences of the protein, a pair of primers was synthesized from the deduced nucleotide sequences of both end amino acids. The lysine-rich protein cDNA was cloned by using the primers to run polymerase chain reaction (PCR) according to a cDNA template from a reverse transcription of mRNA of the plant. The purpose of this studywas to transform lysine and thelysine-rich protein cDNA cloned from this legume into a japonica rice variety, ZH8, and study the possibility of improving the nutritional quality of rice by genetic engineering (Liu et al 1993, Sunet al 1998).

Glycated lysine residues: a marker for non-enzymatic protein glycation in age-related diseases.

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.