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Journal of Inorganic Biochemistry Aug 2024Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been... (Review)
Review
Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes.
Topics: Neuroglobin; Myoglobin; Globins; Nerve Tissue Proteins; Humans; Animals; Heme; Binding Sites; Metalloproteins; Protein Engineering
PubMed: 38759262
DOI: 10.1016/j.jinorgbio.2024.112595 -
PLoS Pathogens Oct 2023Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial...
Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes.
Topics: Humans; Giardia lamblia; Anaerobiosis; Iron-Sulfur Proteins; Mitochondria; Mitochondrial Proteins
PubMed: 37792908
DOI: 10.1371/journal.ppat.1010773 -
Advances in Microbial Physiology 2024Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the... (Review)
Review
Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes. These diverse organelles have lost the major, at one time, definitive components of the mitochondrion (tricarboxylic cycle enzymes and cytochromes), however they all contain the machinery for the assembly of Fe-S clusters, which is the single unifying feature they share. The plasticity of organelles, like the mitochondrion, is therefore evident from its ability to lose its identity as an aerobic energy generating powerhouse while retaining key ancestral functions common to both aerobes and anaerobes. It is interesting to note that the apicoplast, a non-photosynthetic plastid that is present in all apicomplexan protozoa, apart from Cryptosporidium and possibly the gregarines, is also the site of Fe-S cluster assembly proteins. It turns out that in Cryptosporidium proteins involved in Fe-S cluster biosynthesis are localized in the mitochondrial remnant organelle termed the mitosome. Hence, different organisms have solved the same problem of packaging a life-requiring set of reactions in different ways, using different ancestral organelles, discarding what is not needed and keeping what is essential. Don't judge an organelle by its cover, more by the things it does, and always be prepared for surprises.
Topics: Organelles; Mitochondria; Eukaryota; Iron-Sulfur Proteins
PubMed: 38821633
DOI: 10.1016/bs.ampbs.2024.02.001 -
The EMBO Journal Apr 2024Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on...
Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.
Topics: Animals; Humans; Mice; Apoferritins; Cell Lineage; Cytosine; Forkhead Transcription Factors; Iron; T-Lymphocytes, Regulatory
PubMed: 38499786
DOI: 10.1038/s44318-024-00064-x -
Essays in Biochemistry Aug 2023Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox...
Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox reactions, as they are essential components of the engine that life uses to tap into the thermodynamic disequilibria necessary for metabolism. The number of proteins that evolved to catalyze redox reactions is extraordinary, as is the diversification level of metal cofactors and catalytic domain structures involved. Notwithstanding the importance of the topic, the relationship between metals and the redox reactions they are involved in has been poorly explored. This work reviews the structure and function of different prokaryotic organometallic-protein complexes, highlighting their pivotal role in controlling biogeochemistry. We focus on a specific subset of metal-containing oxidoreductases (EC1 or EC7.1), which are directly involved in biogeochemical cycles, i.e., at least one substrate or product is a small inorganic molecule that is or can be exchanged with the environment. Based on these inclusion criteria, we select and report 59 metalloenzymes, describing the organometallic structure of their active sites, the redox reactions in which they are involved, and their biogeochemical roles.
Topics: Oxidoreductases; Metals; Oxidation-Reduction; Metalloproteins; Catalytic Domain
PubMed: 37503682
DOI: 10.1042/EBC20230012 -
Journal of Agricultural and Food... Apr 2024Ferritin nanocages possess remarkable structural properties and biological functions, making them highly attractive for applications in functional materials and... (Review)
Review
Ferritin nanocages possess remarkable structural properties and biological functions, making them highly attractive for applications in functional materials and biomedicine. This comprehensive review presents an overview of the molecular characteristics, extraction and identification of ferritin, ferritin receptors, as well as the advancements in the directional design of high-order assemblies of ferritin and the applications based on its unique structural properties. Specifically, this Review focuses on the regulation of ferritin assembly from one to three dimensions, leveraging the symmetry of ferritin and modifications on key interfaces. Furthermore, it discusses targeted delivery of nutrition and drugs through facile loading and functional modification of ferritin. The aim of this Review is to inspire the design of micro/nano functional materials using ferritin and the development of nanodelivery vehicles for nutritional fortification and disease treatment.
Topics: Ferritins; Structure-Activity Relationship
PubMed: 38518374
DOI: 10.1021/acs.jafc.3c09903 -
Microbiology Spectrum Aug 2023The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for...
The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu or Cd in the cytoplasm, we show that Cu or Cd is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) . This is the first demonstration that Cu affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with and experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases.
Topics: Animals; Cattle; Humans; Xanthine Dehydrogenase; Cadmium; Metalloproteins; Metals
PubMed: 37458582
DOI: 10.1128/spectrum.04814-22 -
Nature Communications Jul 2023Enzymes fold into unique three-dimensional structures to distribute their reactive amino acid residues, but environmental changes can disrupt their essential folding and...
Enzymes fold into unique three-dimensional structures to distribute their reactive amino acid residues, but environmental changes can disrupt their essential folding and lead to irreversible activity loss. The de novo synthesis of enzyme-like active sites is challenging due to the difficulty of replicating the spatial arrangement of functional groups. Here, we present a supramolecular mimetic enzyme formed by self-assembling nucleotides with fluorenylmethyloxycarbonyl (Fmoc)-modified amino acids and copper. This catalyst exhibits catalytic functions akin those of copper cluster-dependent oxidases, and catalytic performance surpasses to date-reported artificial complexes. Our experimental and theoretical results reveal the crucial role of periodic arrangement of amino acid components, enabled by fluorenyl stacking, in forming oxidase-mimetic copper clusters. Nucleotides provide coordination atoms that enhance copper activity by facilitating the formation of a copper-peroxide intermediate. The catalyst shows thermophilic behavior, remaining active up to 95 °C in an aqueous environment. These findings may aid the design of advanced biomimetic catalysts and offer insights into primordial redox enzymes.
Topics: Copper; Biomimetics; Oxidoreductases; Metalloproteins; Amino Acids; Nucleotides
PubMed: 37419896
DOI: 10.1038/s41467-023-39779-6 -
Journal of Agricultural and Food... Dec 2023Ferritin, a distinctive iron-storage protein, possesses a unique cage-like nanoscale structure that enables it to encapsulate and deliver a wide range of biomolecules.... (Review)
Review
Ferritin, a distinctive iron-storage protein, possesses a unique cage-like nanoscale structure that enables it to encapsulate and deliver a wide range of biomolecules. Recent advances prove that ferritin can serve as an efficient 8 nm diameter carrier for various bioinorganic nutrients, such as minerals, bioactive polyphenols, and enzymes. This review offers a comprehensive summary of ferritin's structural features from different sources and emphasizes its functions in iron supplementation, calcium delivery, single- and coencapsulation of polyphenols, and enzyme package. Additionally, the influence of innovative food processing technologies, including manothermosonication, pulsed electric field, and atmospheric cold plasma, on the structure and function of ferritin are examined. Furthermore, the limitations and prospects of ferritin in food and nutritional applications are discussed. The exploration of ferritin as a multifunctional protein with the capacity to load various biomolecules is crucial to fully harnessing its potential in food applications.
Topics: Ferritins; Iron; Minerals; Polyphenols
PubMed: 37955969
DOI: 10.1021/acs.jafc.3c05510 -
Anti-cancer Drugs Jun 2024This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed...
This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed bioinformatics, qPCR, and Western Blot to assess HMOX1 levels in SCLC versus normal tissues and its prognostic relevance. CCK-8, flow cytometry, and thiobarbituric acid assays determined HMOX1's impact on SCLC chemosensitivity, ferroptosis markers, lipid peroxidation, and mic14's role in chemoresistance. In the GSE40275 and GSE60052 cohorts, HMOX1 expression was downregulated in SCLC tissues compared to normal tissues. Higher HMOX1 expression was associated with improved prognosis in the Sun Yat-sen University Cancer Hospital cohort and GSE60052 cohort. The RNA and protein levels of HMOX1 were reduced in drug-resistant SCLC cell lines compared to chemosensitive cell lines. Upregulation of HMOX1 increased chemosensitivity and reduced drug resistance in SCLC, while downregulation of HMOX1 decreased chemosensitivity and increased drug resistance. Upregulation of HMOX1 elevated the expression of ferroptosis-related proteins ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while decreasing the expression of GPX4 and xCT. Conversely, downregulation of HMOX1 decreased the expression of ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while increasing the expression of GPX4 and xCT. Upregulation of HMOX1 promoted cellular lipid peroxidation, whereas downregulation of HMOX1 inhibited cellular lipid peroxidation. Upregulation of HMOX1 reduced the RNA level of mic14, while downregulation of HMOX1 increased the RNA level of mic14. mic14 exhibited inhibitory effects on cellular lipid peroxidation in SCLC cells and contributed to reduced chemosensitivity and increased drug resistance in chemoresistant SCLC cell lines. HMOX1 plays a role in ferroptosis by regulating mic14 expression, thereby reversing chemoresistance in SCLC.
Topics: Humans; Lung Neoplasms; Apoferritins; Heme Oxygenase-1; Ferroptosis; Drug Resistance, Neoplasm; Cell Line, Tumor; Small Cell Lung Carcinoma; RNA; Transferrins
PubMed: 38527419
DOI: 10.1097/CAD.0000000000001588