High Cysteine Content Research Articles

Enhancing high-efficient cadmium biosorption of Escherichia coli via cell surface displaying metallothionien CUP1

ABSTRACT Cadmium (Cd) is one of the common heavy metal pollutants in soil, which can induce various diseases and pose a serious threat to human health. Metallothioneins (MTs) are well-known for their excellent metal binding ability due to a high content of cysteine, which has great potential for heavy metal chelation. In this study, we used the Escherichia coli (E. coli) surface display system LPP-OmpA to construct a recombinant plasmid pBSD-LCF encoding LPP-OmpA-CUP1-Flag fusion protein. Then we displayed the metallothionein CUP1 from Saccharomyces cerevisiae on E. coli DH5α surface for Cd removing. The feasibility of surface display of metallothionein CUP1 in recombinant E. coli DH5α (pBSD-LCF) by Lpp-OmpA system was proved by flow cytometry and western blot analysis, and the specificity of the fusion protein in the recombinant strain was also verified. The results showed that the Cd2+ resistance capacity of DH5α (pBSD-LCF) was highly enhanced by about 200%. Fourier-transform infrared spectroscopy showed that sulfhydryl and sulfonyl groups were involved in Cd2+ binding to cell surface of DH5α (pBSD-LCF). Meanwhile, Cd removal rate by DH5α (pBSD-LCF) was promoted to 95.2%. Thus, the recombinant strain E. coli DH5α (pBSD-LCF) can effectively chelate environmental metals, and the cell surface expression of metallothionein on E. coli can provide new ideas and directions for heavy metals remediation.

The Functional Significance of High Cysteine Content in Eye Lens γ-Crystallins.

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and βγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the βγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens βγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in βB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even β-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

The effectiveness of L-cysteine in the intoxication with the combination of cadmium and lead

The anthropogenic zones formation with an abnormal heavy metals degree negatively affects the safety and quality of livestock products, the health status and animals productivity. Detoxification by complexation with metal-binding proteins and peptides with a high content of cysteine (up to 33%) is one of the intracellular mechanisms of tolerance to heavy metals (HMs). L-cysteine is a natural feed component, practically it does not have any side effects and does not reduce the quality of products, the duration of its use is not limited, while complexing agents cannot be feeded for a long time, as well as the liver and kidneys deseases. The aim of this research was to study the biological effect of the L-cysteine in case of combined chronic cadmium and lead poisoning of white rats in laboratory conditions. Two-month experiment on white rats feeded with a combination of lead and cadmium at the level of 50 and 5 mg/kg respectively were held. It was found that L-cysteine led to the body weight and the function of the central nervous system normalization, the restoration of protein metabolism, and alkaline phosphatase activity, and hematopoietic functions. The feed additive significantly reduced the specific thiolotropic effect of lead and cadmium.

Densely crosslinked protein bioplastics with superior stretchability and desirable wet stability via quality protein restoration

We have demonstrated that dithiol-based crosslinkers are most suitable for protein materials with high contents of cysteine. Crosslinking is always the effective approach to improvement in properties of protein materials. However, most crosslinking technologies damage structures of protein materials due to harsh crosslinking conditions, or form uncontrollable and unstable crosslinkages. In this work, we selected dithiols with different chain lengths and flexibility to form disulfide crosslinkages via reduction and oxidation at room temperature. Sustainable dithiols were screened by computational simulation for bioplastic uses. Since the reactions were performed at room temperature, keratin backbone structures were intact. Our developed model indicates that number of sigma bonds in crosslinkages and retention of crosslinking, dominated by sigma bonds in crosslinkages, quantitatively controlled mechanical properties of regenerated keratin films. Wet-stable keratin films with excellent stretchability were developed without any plasticizers and secondary polymers. Some properties of our bioplastics via quality restoration were similar to some petroleum-based products. For example, feather-derived bioplastics had dry stress, wet stress, dry breaking elongation, and wet elongation of 35 MPa, 10.2 MPa, 70%, and 105%, respectively. After seven days in water, the stress retention of films was as high as 85%. This work promoted the application of keratin bioplastics in food, pharmaceutical and cosmetic industries.

Comparison of cysteine content in whole proteomes across the three domains of life

An empirical observation suggests that Giardia lamblia proteins have larger cysteine content than their counterparts in other organisms. As this parasite lacks conventional antioxidant stress systems, it is generally accepted that high cysteine content helps G. lamblia cope with oxygen toxicity, a strategy apparently shared by other organisms. Here, we question whether the high cysteine content in some organisms is genuine or just a simple assumption based on singular observations. To this end, we analyzed the cysteine content in 78 proteomes of organisms spanning the three domains of life. The results indicate that the cysteine content in eukaryota is approximately double that in archaea and bacteria, with G. lamblia among the highest. Atypical cysteine contents were found in a few organisms correlating with specific environmental conditions, supporting the evolutionary amino acid-level selection of amino acid composition.

Structural Basis of the Immunological Cross-Reactivity between Kiwi and Birch Pollen.

Allergies related to kiwi consumption have become a growing health concern, with their prevalence on the rise. Many of these allergic reactions are attributed to cross-reactivity, particularly with the major allergen found in birch pollen. This cross-reactivity is associated with proteins belonging to the pathogenesis-related class 10 (PR-10) protein family. In our study, we determined the three-dimensional structures of the two PR-10 proteins in gold and green kiwi fruits, Act c 8 and Act d 8, using nuclear magnetic resonance (NMR) spectroscopy. The structures of both kiwi proteins closely resemble the major birch pollen allergen, Bet v 1, providing a molecular explanation for the observed immunological cross-reactivity between kiwi and birch pollen. Compared to Act d 11, however, a kiwi allergen that shares the same architecture as PR-10 proteins, structural differences are apparent. Moreover, despite both Act c 8 and Act d 8 containing multiple cysteine residues, no disulfide bridges are present within their structures. Instead, all the cysteines are accessible on the protein's surface and exposed to the surrounding solvent, where they are available for reactions with components of the natural food matrix. This structural characteristic sets Act c 8 and Act d 8 apart from other kiwi proteins with a high cysteine content. Furthermore, we demonstrate that pyrogallol, the most abundant phenolic compound found in kiwi, binds into the internal cavities of these two proteins, albeit with low affinity. Our research offers a foundation for further studies aimed at understanding allergic reactions associated with this fruit and exploring how interactions with the natural food matrix might be employed to enhance food safety.

Evolution of Cd2+ and Cu+ binding in Helix pomatia metallothioneins.

Metallothioneins (MTs) are small proteins present in all kingdoms of life. Their high cysteine content enables them to bind metal ions, such as Zn2+, Cd2+, and Cu+, providing means for detoxification and metal homeostasis. Three MT isoforms with distinct metal binding preferences are present in the Roman Snail Helix pomatia. Here, we use nuclear magnetic resonance (NMR) to follow the evolution of Cd2+ and Cu+ binding from the reconstructed ancestral Stylommatophora MT to the three H. pomatia MT (HpMT) isoforms. Information obtained from [15N,1H]-HSQC spectra and T2 relaxation times are combined to describe the conformational stability of the MT-metal complexes. A well-behaved MT-metal complex adopts a unique structure and does not undergo additional conformational exchange. The ancestor to all three HpMTs forms conformationally stable Cd2+ complexes and closely resembles the Cd2+-specific HpCdMT isoform, suggesting a role in Cd2+ detoxification for the ancestral protein. All Cu+-MT complexes, including the Cu+-specific HpCuMT isoform, undergo a considerable amount of conformational exchange. The unspecific HpCd/CuMT and the Cu+-specific HpCuMT isoforms form Cu+ complexes with comparable characteristics. It is possible to follow how Cd2+ and Cu+ binding changed throughout evolution. Interestingly, Cu+ binding improved independently in the lineages leading to the unspecific and the Cu+-specific HpMT isoforms. C-terminal domains are generally less capable of coordinating the non-cognate metal ion than N-terminal domains, indicating a higher level of specialization of the C-domain. Our findings provide new insights into snail MT evolution, helping to understand the interplay between biological function and structural features toward a comprehensive understanding of metal preference.

Role of Antimicrobial Peptides in Immunity of Parasitic Leeches

The review summarizes the current state of knowledge about leech immunity, with emphasis on the special role of antimicrobial peptides (AMPs), and highlights the wide variety of primary AMP structures, which seem to correlate with a variety of life strategies and the ecology of ectoparasites. Antimicrobial proteins and AMPs are a diverse class of natural molecules that are produced in all living organisms in response to an attack by a pathogen and are essential components of the immune system. AMPs can have a wide range of antibiotic activities against foreign and opportunistic bacteria, fungi, and viruses. AMPs play an important role in selection of colonizing bacterial symbionts, thus helping multicellular organisms to cope with certain environmental problems. AMPs are especially important for invertebrates, which lack an adaptive immune system. Although many AMPs are similar in physicochemical properties (a total length from 10 to 100 amino acids, a positive total charge, or a high cysteine content), their immunomodulatory activities are specific for each AMP type.

Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3

BackgroundMammalian metallothioneins (MTs) are small (6–7 kDa), intracellular, cysteine-rich, metal-binding proteins involved, inter alia, in the homeostasis of zinc and copper, detoxification of heavy metals, antioxidation against reactive oxygen species, and protection against DNA damage. The high cysteine content (~ 30%) in MTs makes them toxic to bacterial cells during protein production, resulting in low yield. To address this issue, we present for the first time a combinatorial approach using the small ubiquitin-like modifier (SUMO) and/or sortase as fusion tags for high-level expression of human MT3 in E. coli and its purification by three different strategies.ResultsThree different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase recognition motif (LPETG) as removable fusion tags for high-level expression and purification of human MT3 from the bacterial system. In the first strategy, SUMOylated MT3 was expressed and purified using Ulp1-mediated cleavage. In the second strategy, SUMOylated MT3 with a sortase recognition motif at the N-terminus of MT3 was expressed and purified using sortase-mediated cleavage. In the final strategy, the fusion protein His6-SUMO-eSrtA-LPETG-MT3 was expressed and purified by one-step sortase-mediated inducible on-bead autocleavage. Using these three strategies the apo-MT3 was purified in a yield of 11.5, 11, and 10.8 mg/L, respectively, which is the highest yield achieved for MT expression and purification to date. No effect of MT3 on Ni2+-containing resin was observed.ConclusionThe SUMO/sortase-based strategy used as the production system for MT3 resulted in a very high expression level and protein production yield. The apo-MT3 purified by this strategy contained an additional glycine residue and had similar metal binding properties as WT-MT3. This SUMO-sortase fusion system is a simple, robust, and inexpensive one-step purification approach for various MTs as well as other toxic proteins with very high yield via immobilized metal affinity chromatography (IMAC).

In Situ Synthesis of Keratin and Melanin Chromophoric Submicron Particles.

In humans, melanin plays an esthetic role, dictating hair and skin color and traits, while keratin is the protein that comprises most of the epidermis layer. Eumelanin and pheomelanin are types of melanin synthesized from the same building blocks via enzymatic oxidation. Pheomelanin has an additional building block, cysteine amino acid, which affects its final structure. Keratin contains high cysteine content, and by exploiting free thiols in hydrolyzed keratin, we demonstrate the formation of keratin-melanin (KerMel) chromophoric submicron particles. Cryo-TEM analyses found KerMel particle sizes to be 100-300 nm and arranged in the form of a central keratin particle with polymerized l-dopa chains. Attenuated total reflection (ATR)-FTIR, UV-vis, and fluorescence measurements identified new chemical bonds, indicating the formation of KerMel particles. Finally, KerMel replicated natural skin tones and proved cytocompatibility for human epidermal keratinocytes at concentrations below 0.1 mg/mL. Taken together, KerMel is a novel, tunable material that has the potential to integrate into the cosmetic industry.

Roles of Different β-Defensins in the Human Reproductive System: A Review Study.

Human β-defensins (hBDs) are cationic peptides with an amphipathic spatial shape and a high cysteine content. The members of this peptide family have been found in the human body with various functions, including the human reproductive system. Of among β-defensins in the human body, β-defensin 1, β-defensin 2, and β-defensin 126 are known in the human reproductive system. Human β-defensin 1 interacts with chemokine receptor 6 (CCR6) in the male reproductive system to prevent bacterial infections. This peptide has a positive function in antitumor immunity by recruiting dendritic cells and memory T cells in prostate cancer. It is necessary for fertilization via facilitating capacitation and acrosome reaction in the female reproductive system. Human β-defensin 2 is another peptide with antibacterial action which can minimize infection in different parts of the female reproductive system such as the vagina by interacting with CCR6. Human β-defensin 2 could play a role in preventing cervical cancer via interactions with dendritic cells. Human β-defensin 126 is required for sperm motility and protecting the sperm against immune system factors. This study attempted to review the updated knowledge about the roles of β-defensin 1, β-defensin 2, and β-defensin 126 in both the male and female reproductive systems.

BIOINFORMATICS EVALUATION OF CRISP2 GENE SNPs AND THEIR IMPACTS ON PROTEIN

This study evaluated the CRISP2 gene's functional single nucleotide polymorphisms, and its results may be advantageous for future population-based studies and early diagnostic discoveries, particularly in developing effective treatments. The CRISP2 gene encodes a secretory protein with a high cysteine content, which belongs to the family of cysteine-rich secretory proteins (CRISP). SNPs are genetic variations that may affect a protein's structure or functionality. Prior to carrying out a broader population investigation, it is possible to evaluate suspected functional SNPs since it is challenging to uncover functional SNPs in disease-linked genes. As a result, using various bioinformatic prediction models, the potentially harmful three SNPs of the CRISP2 gene were predicted in this in-silico study from the neutral ones. Out of a pool of 260 nsSNPs, three SNPs (L56V, M176I, and C196R) been selected to anticipate their impacts on functions and structures along with their capability to impair protein stability. Actually, two of the three SNPs in the CRISP2 gene L56V and C196R were identified as possibly detrimental, although M176I was not. But all of these SNPs dropped significantly protein stability, per the I-Mutant suite.

Amino acid content in spring common wheat grain

The aim of the study was to determine the differences in the content of amino acids in wheat grains of the world collection of VIR in the agro-climatic conditions of the Republic of Tatarstan and to search for sources of increased content of essential amino acids for the use of these data in breeding. The study was conducted in 2021-2022 at the experimental base of the Tatar Research Institute of Agriculture of the Kazan Scientific Center of the Russian Academy of Sciences, located 15 km away from Kazan, the soil of the experimental site is gray forest. 354 samples of spring wheat were studied. The determination of the content of seventeen amino acids and protein in wheat grain was carried out using the near-infrared spectroscopy method on the DS 2500F device from FOSS. The protein content in the grain has a significant effect on the amount of essential acids. The correlation between the protein content and the amount of essential acids in wheat grain is high in 2021. r = 0.91 ± 0.02 and average in 2022. r = 0.69± 0.04. The highest content of glutamine in wheat grain was noted - up to 2.22 % and proline - up to 1.55 %. The source of high grain content of histidine 0.38- 0.40 %, isoleucine 0.53-0.54 %, lysine 0.61-0.62 %, methionine 0.28-0.30 %, phenylalanine 0.66-0.67 %, threonine 0.52-0.56 % and tyrosine 0.31-0.36 % was the Ranee sample (k-26982). The Panifor sample was a source of high grain content of cysteine 0.58-0.63 %, histidine 0.39 % and threonine 0.51-0.58 %. The Arrino sample (k-65995) is a source of high grain content of leucine 0.82-0.91 %, tyrosine 0.31-0.34 % and valine 0.75-0.78 %. According to the lysine content in protein (grams of amino acid per 100 grams of protein), samples Ranee (k-26982) - 3.63-3.66 and NIL Avocet Yr15 (k-66241) - 2.86-4.52 were isolated. According to the content of methionine in the protein, samples of Uffo (k- 66798) - 1.80-1.8 and Fiorina (k-66034) - 1.70-1.91 were isolated. According to the threonine content, three samples Ranee (k-26982) - 3.04-3.36, Panifor (k-66039) - 3.09-3.20 and Cub (k- 62510) - 3.07-3.20 were distinguished. The source of the high tryptophan content in the protein were samples Ethos (k- 65002) - 2.00-2.27, Tulaykovskaya 108 (k- 65452) - 2.01-2.26 and Ekada 109 (k-66239) - 2.07-2.14.

Structural Role of Cadmium and Zinc in Metallothionein Oxidation by Hydrogen Peroxide: The Resilience of Metal-Thiolate Clusters.

Oxidative stress is a state involving an imbalance of reactive oxygen species in a cell and is linked to a variety of diseases. The metal-binding protein metallothionein (MT) may play a role in protection due to its high cysteine content. Many studies have shown that oxidative stress will cause MT to both form disulfide bonds and release bound metals. However, studies on the more biologically relevant partially metalated MTs have been largely neglected. Additionally, most studies to date have used spectroscopic methods that cannot detect specific intermediate species. In this paper, we describe the oxidation and the subsequent metal displacement pathway of fully and partially metalated MTs with hydrogen peroxide. The rates of the reactions were monitored using electrospray ionization mass spectrometry (ESI-MS) techniques, which resolved and characterized the individual intermediate Mx(SH)yMT species. The rate constants were calculated for each species formation. Through ESI-MS and circular dichroism spectroscopy, it was found that the three metals in the β-domain were the first to be released from the fully metalated MTs. The Cd(II) in the partially metalated Cd(II)-bound MTs rearranged to form a protective Cd4MT cluster structure upon exposure to oxidation. The partially metalated Zn(II)-bound MTs oxidized at a faster rate as the Zn(II) did not rearrange in response to oxidation. Additionally, density functional theory calculations showed that the terminally bound cysteines were more negative and thus more susceptible to oxidation than the bridging cysteines. The results of this study highlight the importance of metal-thiolate structures and metal identity in MT's response to oxidation.

Plant Protein Can Be as Efficient as Milk Protein to Maintain Fat Free Mass in Old Rats, Even When Fat and Sugar Intakes Are High

BackgroundAlternative, sustainable, and adequate sources of protein must be found to meet global demand. ObjectivesOur aim was to assess the effect of a plant protein blend with a good balance of indispensable amino acids and high contents of leucine, arginine, and cysteine on the maintenance of muscle protein mass and function during aging in comparison to milk proteins and to determine if this effect varied according to the quality of the background diet. MethodsOld male Wistar rats (n = 96, 18 mo old) were randomly allocated for 4 mo to 1 of 4 diets, differing according to protein source (milk or plant protein blend) and energy content (standard, 3.6 kcal/g, with starch, or high, 4.9 kcal/g, with saturated fat and sucrose). We measured: every 2 mo, body composition and plasma biochemistry; before and after 4 mo, muscle functionality; after 4 mo, in vivo muscle protein synthesis (flooding dose of L-[1-13C]-valine) and muscle, liver, and heart weights. Two-factor ANOVA and repeated measures 2-factor ANOVA were conducted. ResultsThere was no difference between protein type on the maintenance during aging of lean body mass, muscle mass, and muscle functionality. The high-energy diet significantly increased body fat (+47%) and heart weight (+8%) compared to the standard energy diet but had no effect on fasting plasma glucose and insulin. Muscle protein synthesis was significantly stimulated by feeding to the same extent in all groups (+13%). ConclusionsSince high-energy diets had little impact on insulin sensitivity and related metabolism, we could not test the hypothesis that in situations of higher insulin resistance, our plant protein blend may be better than milk protein. However, this rat study offers significant proof of concept from the nutritional standpoint that appropriately blended plant proteins can have high nutritional value even in demanding situations such as aging protein metabolism.

Characterization of the immunomodulatory protein isolated from guduchi (Tinospora cordifolia) churna, an ayurvedic rejuvenator

Objective: Guduchi [Tinospora cordifolia (Willd.) f. and Thoms.], a rasayana (rejuvenator) in the traditional system of Indian medicine (Ayurveda), is known for its diverse bioactivities. The aim of this study was to isolate the immunomodulatory protein (ImP) from commercial guduchi churna (stem powder) and characterize it. Methods: Guduchi churna extract following 80% ammonium sulfate precipitation was chromatographed on Q-Sepharose. Purified guduchi ImP was evaluated for its lymphoproliferative and macrophage activation properties, structural relatedness to certain plant lectins, amino acid composition, stability to trypsin, and N-terminal sequencing. Results: The purified protein, obtained in a yield of 140[Formula: see text]mg/100[Formula: see text]g guduchi churna, showed a molecular mass of [Formula: see text] kDa by SDS-PAGE. Guduchi ImP (10 [Formula: see text]g) showed seven-fold increase over control in mitogenic activity of murine splenocytes, and a four-fold increase over control in nitric oxide (NO) release from murine peritoneal exudates cells (PECs). These activities were abolished in pronase-treated guduchi ImP. Guduchi ImP does not agglutinate rabbit erythrocytes. Rabbit antiserum to guduchi stem ImP strongly reacted with purified guduchi ImP, and did not recognize common plant lectins (concanavalin A (Con A), poke weed mitogen (PWM) and garlic lectin). Amino acid composition of guduchi ImP shows high content of acidic amino acids, serine (12.4%) and cysteine (10.2%); guduchi ImP is highly stable to trypsin and has a blocked N-terminus. Conclusion: Guduchi ImP is an acidic, non-lectin ImP with a high content of serine and cysteine, and appears to have an immense potential for future structural, immunological, and therapeutic studies.

Increase in cysteine-mediated multimerization under attractive protein–protein interactions

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein–protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein–protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.

Wool keratin as a novel alternative protein: A comprehensive review of extraction, purification, nutrition, safety, and food applications.

The growing global population and lifestyle changes have increased the demand for specialized diets that require protein and other essential nutrients for humans. Recent technological advances have enabled the use of food bioresources treated as waste as additional sources of alternative proteins. Sheep wool is an inexpensive and readily available bioresource containing 95%-98% protein, making it an outstanding potential source of protein for food and biotechnological applications. The strong structure of wool and its indigestibility are the main hurdles to achieving its potential as an edible protein. Although various methods have been investigated for the hydrolysis of wool into keratin, only a few of these, such as sulfitolysis, oxidation, and enzymatic processes, have the potential to generate edible keratin. In vitro and in vivo cytotoxicity studies reported no cytotoxicity effects of extracted keratin, suggesting its potential for use as a high-value protein ingredient that supports normal body functions. Keratin has a high cysteine content that can support healthy epithelia, glutathione synthesis, antioxidant functions, and skeletal muscle functions. With the recent spike in new keratin extraction methods, extensive long-term investigations that examine prolonged exposure of keratin generated from these techniques in animal and human subjects are required to ascertain its safety. Food applications of wool could improve the ecological footprint of sheep farming and unlock the potential of a sustainable protein source that meets demands for ethical production of animal protein.

Human hair proteins as natural reactive oxygen species scavengers for in vitro applications.

Human hair proteins are recognized for their intrinsically high cysteine content. They can be solubilized while preserving their highly reductive thiol groups for free radical scavenging applications. The presence of aromatic and nucleophilic amino acids such as methionine, serine, phenylalanine, and threonine further contribute to the antioxidative potential of this material. Herein, utilizing the DPPH (2,2-diphenyl-1-picrylhydrazyl) and acellular 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) assays, keratins are demonstrated to possess the highest radical scavenging activity among the studied hair proteins. Consequently, protection against hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs) cultured in human hair keratin supplemented media is demonstrated. Quenching of reactive oxygen species in the HDF is observed using the CellROX Green dye and the expression levels of antioxidant (HMOX1, SOD2, GPX1) and tumor suppressor (TP53) genes is analyzed using qPCR. Collectively, this study presents further evidence and demonstrates the in vitro application potential of hair proteins, especially keratins, as an antioxidizing supplement.

Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1.

Cells that experience high levels of oxidative stress respond by inducing antioxidant proteins through activation of the protein transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is negatively regulated by the E3 ubiquitin ligase Kelch-like ECH-associated protein 1 (Keap1), which binds to Nrf2 to facilitate its ubiquitination and ensuing proteasomal degradation under basal conditions. Here, we studied protein folding and misfolding in Nrf2 and Keap1 in yeast, mammalian cells, and purified proteins under oxidative stress conditions. Both Nrf2 and Keap1 are susceptible to protein misfolding and inclusion formation upon oxidative stress. We propose that the intrinsically disordered regions within Nrf2 and the high cysteine content of Keap1 contribute to their oxidation and the ensuing misfolding. Our work reveals previously unexplored aspects of Nrf2 and Keap1 regulation and/or dysregulation by oxidation-induced protein misfolding.