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FEBS Open Bio Jun 2024Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral,...
Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric-type class II chelatase which is able to use not only Ni as a physiological metal substrate, but also Co as a nonphysiological substrate with higher activity than for Ni. The Ni/Co-chelatase function found in CfbA is also observed in SirB, a descendant, monomeric-type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni-chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni-chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni-chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni-binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni-chelatase reaction in a class II chelatase architecture.
PubMed: 38923868
DOI: 10.1002/2211-5463.13849 -
Animal Science Journal = Nihon Chikusan... 2024We hypothesized that teats with a teat apex score (TAS) of 4 on a 4-point scale would exhibit elevated levels of denatured collagen compared with teats with lower TAS....
We hypothesized that teats with a teat apex score (TAS) of 4 on a 4-point scale would exhibit elevated levels of denatured collagen compared with teats with lower TAS. We procured keratin layer and smooth muscle samples from Holsteins with TAS ranging from 1 to 4, as well as from crossbred heifers (Japanese Black male and Holstein female) with TAS of 1. Teats with a TAS of 4 demonstrated increased total collagen content, higher amounts of type I collagen (the harder, thicker variant), and reduced amounts of type III collagen (the softer, thinner variant) compared with teats with lower TAS. Teats with TAS of 3 and 4 exhibited evidence of damaged collagen in smooth muscle layers compared with teats with TAS of 1. Additionally, we identified 47-kDa heat shock protein-positive fibroblasts in the smooth muscles of teats with TAS of 3 and 4. Therefore, the smooth muscle of teats with a TAS of 4 exhibited increased amounts of denatured collagen in comparison to teats with lower TAS.
Topics: Animals; Cattle; Female; Muscle, Smooth; Collagen; Keratins; Mammary Glands, Animal; Protein Denaturation; Male; Collagen Type I; Fibroblasts; Collagen Type III
PubMed: 38923230
DOI: 10.1111/asj.13969 -
Acta Chimica Slovenica Apr 2024The aim of this study is to optimize the extraction process and characterize the proteins found in fenugreek seeds. The water and oil holding capacities, coagulated...
The aim of this study is to optimize the extraction process and characterize the proteins found in fenugreek seeds. The water and oil holding capacities, coagulated protein content, foaming and emulsification properties of the isolated proteins at all extraction conditions were investigated. Also, solubility, molecular weights, structural and thermal properties were determined. In the extraction processes carried out at different pHs (pH 6.0-12.0) and solid:solvent ratios (20-60 g/L), it was determined that the highest extraction yield (94.3±0.3%) was achieved when the pH was 11.47 and the solid-solvent ratio was 34.50 g/L. Three distinct bands (46, 59 and 80 kDa) in the range of 22-175 kDa were determined for the fenugreek seed protein isolate obtained at optimum extraction conditions. Protein secondary structures were achieved using Fourier Transform Infrared (FT-IR) spectra and it was determined that β-sheet structures were highly present. In addition, denaturation temperatures and denaturation enthalpy were calculated as ~119°C and 28 mJ/g, respectively.
Topics: Trigonella; Seeds; Plant Proteins; Hydrogen-Ion Concentration; Spectroscopy, Fourier Transform Infrared; Solubility; Molecular Weight
PubMed: 38919106
DOI: 10.17344/acsi.2023.8576 -
BioRxiv : the Preprint Server For... Jun 2024Electrolytes are essential parts of the environment for all life forms, where proteins, water, and solutes interplay to support vital activities. However, a fundamental...
Electrolytes are essential parts of the environment for all life forms, where proteins, water, and solutes interplay to support vital activities. However, a fundamental understanding of the effect of ionic solutes on proteins remains elusive for more than a century. Here we show how some ionic solutes can serve as potent denaturants despite the absence of direct protein-ion interactions. We demonstrate dramatic differences between denaturation potency of different ionic solutes with lithium bromide (LiBr) being the strongest denaturant and sodium bromide (NaBr) being the least potent. Experiments and simulations indicate the presence of certain ions disrupts the structure of water network, thereby induce protein denaturation indirectly via an entropy-driven mechanism. We further introduce a scalable strategy for protein waste revalorization, distinguished by the closed-loop recycling of denaturants, straightforward protein separation, and facile manufacturing, all enabled by the entropy-driven denaturation by LiBr. Through successful isolation and systematic study of indirect solute effects, our findings suggest a unified and generally applicable framework for decoding of the protein-water-solute nexus, where all current studies can be easily incorporated. Besides, our regeneration approach underscores the feasibility of repurposing protein waste into valuable biomaterials in a sustainable way with wide-reaching application potential.
PubMed: 38915630
DOI: 10.1101/2024.06.12.598657 -
International Journal of Biological... Jun 2024Understanding how shear affects whey protein stability is crucial to deal with typical industrial issues occurring at the bulk solution/surface interface, such as...
Understanding how shear affects whey protein stability is crucial to deal with typical industrial issues occurring at the bulk solution/surface interface, such as fouling during heat treatments. However, at the state of the art, this effect remains unclear, contrary to that of temperature. This article presents a novel strategy to study the impact of shear rate and concentration on the accumulation of whey protein surficial deposits. It consists in applying a range of shear rates (0-200 s) at controlled temperature (65 °C) on whey protein solutions (5-10 wt%) by a parallel plate rheometer equipped with a glass disc, thus allowing the off-line characterization of the deposits by microscopy. Our results highlight an unequivocal effect of increasing shear stress. At 5 wt%, it fosters the formation of primary deposits (≈ 10 μm), whereas at 10 wt% it results in the development of complex branched structures (≈ 50 μm) especially for shear rates ranging from 140 s to 200 s. Based on the classification by size of the observed populations, we discuss possible hypotheses for the deposit growth kinetics, involving the interplay of different physico-chemical protein-surface interactions and paving the way to future further investigations.
PubMed: 38908625
DOI: 10.1016/j.ijbiomac.2024.133291 -
Methods in Molecular Biology (Clifton,... 2024We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass...
We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.
Topics: Proteomics; Single-Cell Analysis; Chromatography, Liquid; Humans; Mass Spectrometry; Proteome; Proteins
PubMed: 38907148
DOI: 10.1007/978-1-0716-3934-4_7 -
Molecular Biology Reports Jun 2024Protein nanocages resemble natural biomimetic carriers and can be engineered to act as targeted delivery systems, making them an attractive option for various drug...
Protein nanocages resemble natural biomimetic carriers and can be engineered to act as targeted delivery systems, making them an attractive option for various drug delivery and biomedical applications. Our research investigated the genetic link of a specific anti-HER2 peptide (LTVSPWY) to the exposed N-terminal region of the maize (Zea mays) ferritin 1 (ZmFer1) protein nanocage, employing either a 7-amino acid (for LTVS-ZmFer1) or 16-amino acid (for LTVS-L-ZmFer1) linker. We utilized a heat treatment method to load the chemotherapeutic drug doxorubicin into the protein nanocage. The construct with the longer linker (LTVS-L) produced a greater amount of soluble protein nanocage and was selected for further experiments. The average size, polydispersity index, and zeta potential of the engineered protein nanocage were 19.01 nm, 0.168, and - 2.13 mV, respectively. The LTVS-L-ZmFer1 protein nanocage exhibited excellent thermal stability, withstanding temperatures up to 100 °C with only partial denaturation. Furthermore, we observed that cellular uptake of the LTVS-L-ZmFer1 protein nanocages in HER2-positive breast cancer cells was significantly higher compared to ZmFer1 after labeling with FITC (fluorescein isothiocyanate) (P-value = 0.0001). In addition, we observed a significant decrease in the viability of SKBR3 cells when treated with DOX-loaded LTVS-L-ZmFer1 protein nanocages compared to cells treated with DOX-loaded ZmFer1 protein nanocages. Therefore, this new treatment strategy may prove to be an effective way to reduce both the side effects and toxicity associated with conventional cancer treatments in patients with HER2-positive breast cancer.
Topics: Humans; Receptor, ErbB-2; Doxorubicin; Drug Delivery Systems; Cell Line, Tumor; Breast Neoplasms; Recombinant Proteins; Ferritins; Zea mays; Protein Engineering; Female; Drug Carriers; Plant Proteins
PubMed: 38904710
DOI: 10.1007/s11033-024-09636-w -
Frontiers in Nutrition 2024Biocatalysts (enzymes) play a crucial role in catalyzing specific reactions across various industries, often offering environmentally friendly and sustainable...
BACKGROUND
Biocatalysts (enzymes) play a crucial role in catalyzing specific reactions across various industries, often offering environmentally friendly and sustainable alternatives to chemical catalysts. However, their catalytic activities are susceptible to denaturation. In this study, we present the discovery of novel protein-based biocatalysts derived from processed foods, including skimmed milk, soy milk, cheese, and dried tofu. These food catalysts exhibit high availability, low cost, safety, and thermo-stability.
RESULTS
Focusing on the physiologically intriguing coenzyme pyrroloquinoline quinone (PQQ), we observed that the reaction with glycine to form imidazolopyrroquinoline (IPQ) did not proceed efficiently when PQQ was present at very low concentrations. Surprisingly, in the presence of protein-based foods, this reaction was significantly accelerated. Notably, skimmed milk enhanced the PQQ detection limit (600 times lower) during high-performance liquid chromatography (HPLC) following IPQ derivatization. Milk appears to facilitate the reaction between PQQ and various amino acids, primary amines, and secondary amines. Further investigations revealed that food catalysis operates through a non-enzymatic mechanism. Additionally, nuclear magnetic resonance spectroscopy demonstrated that milk components interacted with amino substrates due to the ability of amines to react with quinones on colloidal surfaces.
CONCLUSION
These practical food catalysts not only contribute to environmental safety but also hold significance across diverse scientific domains. Non-enzymatic protein catalysts find applications in biocatalysis, organic synthesis, food technology, analytical chemistry, and fundamental nutritional and evolutionary studies.
PubMed: 38903631
DOI: 10.3389/fnut.2024.1391681 -
Circulation Research Jun 2024Cardiac fibroblast activation contributes to adverse remodeling, fibrosis, and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-β...
BACKGROUND
Cardiac fibroblast activation contributes to adverse remodeling, fibrosis, and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-β (transforming growth factor-β)/Smad (small mother against decapentaplegic)-3 activation protects the pressure-overloaded heart by preserving the matrix, sustained TGF-β activation is deleterious, accentuating fibrosis and dysfunction. Thus, endogenous mechanisms that negatively regulate the TGF-β response in fibroblasts may be required to protect from progressive fibrosis and adverse remodeling. We hypothesized that Smad7, an inhibitory Smad that restrains TGF-β signaling, may be induced in the pressure-overloaded myocardium and may regulate fibrosis, remodeling, and dysfunction.
METHODS
The effects of myofibroblast-specific Smad7 loss were studied in a mouse model of transverse aortic constriction, using echocardiography, histological analysis, and molecular analysis. Proteomic studies in S7KO (Smad7 knockout) and overexpressing cells were used to identify fibroblast-derived mediators modulated by Smad7. In vitro experiments using cultured cardiac fibroblasts, fibroblasts populating collagen lattices, and isolated macrophages were used to dissect the molecular signals responsible for the effects of Smad7.
RESULTS
Following pressure overload, Smad7 was upregulated in cardiac myofibroblasts. TGF-β and angiotensin II stimulated fibroblast Smad7 upregulation via Smad3, whereas GDF15 (growth differentiation factor 15) induced Smad7 through GFRAL (glial cell line-derived neurotrophic factor family receptor α-like). MFS7KO (myofibroblast-specific S7KO) mice had increased mortality, accentuated systolic dysfunction and dilative remodeling, and accelerated diastolic dysfunction in response to transverse aortic constriction. Increased dysfunction in MFS7KO hearts was associated with accentuated fibrosis and increased MMP (matrix metalloproteinase)-2 activity and collagen denaturation. Secretomic analysis showed that Smad7 loss accentuates secretion of structural collagens and matricellular proteins and markedly increases MMP2 secretion. In contrast, Smad7 overexpression reduced MMP2 levels. In fibroblasts populating collagen lattices, the effects of Smad7 on fibroblast-induced collagen denaturation and pad contraction were partly mediated via MMP2 downregulation. Surprisingly, MFS7KO mice also exhibited significant macrophage expansion caused by paracrine actions of Smad7 null fibroblasts that stimulate macrophage proliferation and fibrogenic activation. Macrophage activation involved the combined effects of the fibroblast-derived matricellular proteins CD5L (CD5 antigen-like), SPARC (secreted protein acidic and rich in cysteine), CTGF (connective tissue growth factor), ECM1 (extracellular matrix protein 1), and TGFBI (TGFB induced).
CONCLUSIONS
The antifibrotic effects of Smad7 in the pressure-overloaded heart protect from dysfunction and involve not only reduction in collagen deposition but also suppression of MMP2-mediated matrix denaturation and paracrine effects that suppress macrophage activation through inhibition of matricellular proteins.
PubMed: 38899461
DOI: 10.1161/CIRCRESAHA.123.323360 -
Protein Science : a Publication of the... Jul 2024Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing...
Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (K) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of K tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had K values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's K remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.
Topics: Humans; Zymomonas; Pyruvate Kinase; Amino Acid Substitution; Protein Stability; Bacterial Proteins; Enzyme Stability; Liver; Phosphoenolpyruvate
PubMed: 38895978
DOI: 10.1002/pro.5075