The Cytb561 family of transmembrane proteins typically contain six transmembrane α-helix domains (51–53). Sequence alignment indicates that all retrieved sequences exhibit four completely conserved sequences located in the second to fifth transmembrane segments (14). Four consecutive central helices form the core domain of CYB561 (Fig. 1). The core of the four helices comprises two pairs of histidine (His) residues (14,51); the first and third residues are located on the cytoplasmic side, while the second and fourth are located at the boundary region of the outer (or intravesicular) hydrophilic ring and transmembrane helix (54–56). The CYB561 core domain, via its four His residues, coordinates to heme-b groups on both sides of the membrane (57–59). The core domain facilitates intramolecular electron transfer by accepting electrons from ASC (60). It is structurally similar to other redox domains, such as dopamine β-monoxygenase redox domains and the dopamine β-monooxygenase N-terminal (DOMON) domain (24), and can be a component of other proteins. The basic characteristics of hCytb561 family members, including their genomic locations and sequence sizes, are shown in Table I. They are divided into different categories according to their specific motifs (Fig. 1). CYB561, Dcytb and LCytb have been assigned to the animals/neuroendocrine class due to presence of motif 1 [FN(X)HP(X)₂M(X)₂G(X)₅G(X)ALLVYR] and motif 2 [YSLHSW(X)G] in their core structures (15). 101F6 and its analogs have been classified in the animals/TSF class due to the presence of modified motif 1 [LFSWHP(X)₂M(X)₃F(X)₃M(X)EAIL(X)SP(X)₂SS] in their structures. SDR2 belongs to the SDR2 class due to the presence of three DOH domains or a DOMON domain (14), which is homologous to a domain found in dopamine-β-hydroxylase (61). All family members have transmembrane electron transfer activity or contain at least two heme groups that may contribute to the integrity or binding of the transmembrane protein structure (56). The double-electrode voltage-clamp technique has demonstrated the ability of SDR2 to conduct electric current (62).

Human CYB561 is estimated to lack the first 22 amino acids at the N-terminal of the cytoplasmic side predicted for the bovine cell sequence. Srivastava et al (63) hypothesized that the gene product of human CYB561 contains five transmembrane helical structures. The crystal structure of Arabidopsis thaliana Cytb561 suggests that this protein may function as a dimer (13). Additionally, two highly conserved amino acids, Lys-81 and His-106, have been demonstrated to be important in substrate recognition and catalysis (64). To illustrate the structures of hCytb561 family members, data on their 3-dimensional structures have been obtained from the UniProt (Universal Protein Resource) website (https://www.uniprot.org/uniprotkb) and are presented in Fig. 2.

The Cytb561 family uses ASC, an extracapsular electron donor, on one side of the membrane to provide electrons, catalyze transmembrane electron transport, and reduce monodehydroascorbate (MDA), a partially oxidized form of ASC that acts as an intracapsular electron receptor, thereby facilitating the regeneration of ASC (64,65). Motif 2 was originally thought to be an MDA free-radical-binding sequence (60). The reduced expression of the CYB561 homologue in Drosophila memory mutant (nemy) has been shown to lead to defective memory retention, confirming the role of Cytb561 in memory retention and its influence on neural function (66). In addition, the role of Cytb561 in ASC regeneration has been confirmed by the fact that ASC in erythrocytes can reduce extracellular MDA (67,68). ASC has been confirmed to function as an electron donor for mouse DCytb (67). Furthermore, B-type cytochromes with biophysical properties similar to those of Cytb561 from bovine chromaffin granules have been reported to reduce ASC in the cell membranes of certain plants (69,70).

In vitro, all Cytb561 members exhibit Fe³⁺ reductase activity (71). DCytb in the cell membranes of mouse duodenal epithelial cells has been shown to reduce intestinal Fe³⁺ and thereby facilitate the uptake of iron, which ameliorates hypoxia and iron deficiency (17). In addition, the increased expression of DCytb in mice with total iron overload (hemochromatosis) promotes iron uptake (72). The transmembrane Fe (3+) reductase activity of CYB561 has been demonstrated in ASC-loaded protein liposomes (57). Furthermore, DCytb uses cytoplasmic ASC as an electron donor to reduce extracellular Fe³⁺ to Fe²⁺ in rabbit peritoneal neutrophils (73). Three members of the Cytb561 family, namely mouse Cytb561 and mouse and Drosophila SDR2, have been found to have iron reductase activity (71). These findings indicate that when free Fe³⁺ and ASC are in the same membrane compartment, Fe³⁺ easily obtains electrons from ASC and is reduced to Fe²⁺, which can generate free radicals via the Fenton reaction. However, when Fe³⁺, ASC and Cytb561 are separated, the ASC-mediated reduction of Fe³⁺ may be moderated. Whether Cytb561 family members reduce Fe³⁺or MDA in vivo is likely to depend on the availability of the substrate and the biological environment.

First identified in chromaffin vesicles that synthesize catecholamines, CYB561 was later confirmed to be present in neuroendocrine tissues (74). RNA sequencing has shown that CYB561 is widely expressed in eight systems (locomotion, digestive, respiratory, circulatory, urinary, reproductive, endocrine and nervous systems) of the normal human body. According to gene expression data from the Bgee database (https://bgee.org), the CYB561 gene is expressed in the adrenal gland and 223 other tissues. Pathologically, CYB561 is expressed in HeLa cervical cancer cells and melanoma cells (74). In addition, its mRNA levels are significantly upregulated in SW480 colon cancer, HL-60 T-cell lymphoma and K-562 chronic myeloid leukemia cells but not in Burkitt's lymphoma (75). Under physiological conditions, CYB561 in natural chromaffin granules, vesicles and recombinant membrane systems exhibits ‘electron shuttle’ activity between ASC and membrane permeable ferricyanide or MDA (57,76), which is associated with cell metabolism and mitochondrial activation (77) (Fig. 3), and affects heart rate and blood pressure through regulation of the adrenaline pathway (78).

Pathogenic homozygous mutations in the CYB561 gene have been reported to lead to ASC deficiency in catecholamine-secreting vesicles and functional dopamine b-hydroxylase deficiency, resulting in orthostatic hypotension syndrome (78). In CYB561^(−/-) mice, it was found that the concentration of norepinephrine in whole brain homogenate and adrenal glands was reduced compared with that in wild-type mice (78). CYB561 is also involved in the progression of certain tumors and influences their prognosis. For example, a low expression level of CYB561 mRNA was found to be associated with a poor prognosis in patients with ovarian cancer, suggesting that CYB561 may be a single-gene prognostic biomarker (79). In addition, in another study CYB561 was demonstrated to promote the growth and metastatic potential of castration-resistant neuroendocrine prostate cancer (80). Our previous study found that CYB561 expression was upregulated in breast cancer; associated with human epidermal growth factor receptor 2 (HER2), immune cells, histological grade and molecular subtypes; and associated with the poor prognosis of patients (81), which is consistent with other results reported in the literature (82). Moreover, our previous study demonstrated that CYB561 promotes the proliferation, migration and invasion of breast cancer cells and inhibits apoptosis (83). The knockdown of CYB561 changes the Fe²⁺ and total iron content of cancer cells and the expression of the iron absorption and transport-related proteins TFRC and DMT1, indicating that it is involved in the iron metabolism of breast cancer cells (83). In addition, recent literature shows that CYB561 plays a role in promoting the proliferation of HER2-positive breast cancer cells by inhibiting the degradation of H2A histone family member Y (84).

Dcytb is a plasma membrane protein with 40–50% homology to CYB561 (17). The predicted ASC- and DHA-binding motifs in the Cytb561 family are highly conserved in Dcytb (17) (Fig. 3). The gene encoding Dcytb is widely expressed in human tissues and highly expressed in the duodenal brush border membrane. Dcytb serves as the plasma membrane iron reductase of mammals, facilitating the absorption of dietary iron at the brush edge of duodenal cells by reducing Fe³⁺ to Fe²⁺ and cooperating with DMT1 to transport Fe²⁺ to cells in the small intestine (21) (Fig. 3). It also participates in the regeneration of extracellular ASC (85) by reducing extracellular MDA (86), and may also be a transmembrane reductase of copper (87). The inactivation of the L-gulono-g-lactone oxidase (GULO) gene under physiological conditions prevents the synthesis of the protein GULO enzyme and therefore ASC synthesis. Dcytb can reduce and regenerate oxidized ASC while reducing Fe(3+) to Fe(2+), thereby promoting iron absorption. Therefore, it is speculated that the demand for this reductase increases when the demand for iron absorption is strong (88).

Under normoxic conditions, hemoglobin in the reticulocytes of CYBRD1^(−/-) mice is impaired, and under hypoxic conditions, these mice exhibit reduced duodenal reductase activity and spleen iron reserves, leading to abnormal erythropoiesis (89). As no evidence of hypoxia-induced iron reductase activity has been observed in the CYBRD1^(−/-) mice, this suggests that Dcytb may be the only hypoxia-induced iron reductase in the duodenum. Hypoxia has been demonstrated to strongly upregulate Dcytb expression through a hypoxia-inducible factor-2α (HIF-2α)-dependent mechanism. This activation of HIF-2α also upregulates DMT1 expression, which disrupts local iron homeostasis and promotes cell proliferation, which contributes to the development of colon cancer (90) (Fig. 3). In addition, the upregulation of the iron-import-related proteins Dcytb, DMT1 and TFRC in colorectal cancer cells is associated with increased intracellular iron content (91). An analysis of data from The Cancer Genome Atlas (TCGA) database revealed that the expression of CYBRD1 is upregulated in ovarian cancer (92), and its increased expression is associated with a reduction in overall survival times (92). In another study it was shown that CYBRD1 is upregulated in glioma cell lines, and its silencing reduces the aggressiveness of glioma cells, indicating its potential as a biomarker for glioma recurrence (93) (Fig. 3). Boult et al (94) reported that the expression of CYBRD1 is upregulated during the progression of Barrett's esophagus to adenocarcinoma, suggesting its involvement in the development of esophageal adenocarcinoma through increased iron absorption. In addition, tumor-initiating cells from the breast and prostate origin have been found to exhibit the altered expression of various genes associated with iron metabolism, including CYBRD1, endothelial PAS domain-containing protein 1/HIF-2α and TFRC (95). By contrast, CYBRD1 has also been reported to exhibit tumor-inhibiting effects. Analysis of gene expression in a Gene Expression Omnibus dataset showed that CYBRD1 expression is downregulated in bladder cancer compared with normal bladder mucosa, suggesting that it may have a tumor-inhibitory role (96). Dcytb has been shown to be an important predictor of prognosis in patients with breast cancer, with high Dcytb expression serving as an overall predictor of distant metastasis-free survival and exhibiting a significant association with increased relapse-free survival (23). However, the role of Dcytb in breast cancer cells was indicated to be unrelated to iron metabolism; instead it was demonstrated to delay tumor progression by inhibiting the activation of focal adhesion kinase, a kinase that plays a central role in tumor cell adhesion and metastasis (23). In addition, analysis of TCGA data has shown that the CYBRD1 protein level decreases with increasing lung adenocarcinoma stage and grade, indicating a negative correlation between CYBRD1 expression and the progression of lung adenocarcinoma. Moreover, the low expression of CYBRD1 is associated with poor overall and disease-specific survival (97).

This reductase is present in the late endolysosome-lysosome membrane; it is expressed at high levels in the adrenal gland, lymph nodes, B lymphocytes and monocytes, but is downregulated in sepsis (98) and skin warts caused by human papillomavirus infection (99).

LCytb demonstrates iron reductase activity in yeast (67,75). It also participates in iron homeostasis in endosomes and lysosomes. In late endosomes and lysosomes, LCytb uses ASC as an electron donor to reduce Fe³⁺ to Fe²⁺, which is then transferred by DMT1 into the cytoplasm. In addition, LCytb regulates the iron cycle in macrophages by reducing the expression of lysosomal reductase, which is part of the innate immune response that prevents the proliferation of pathogens and sepsis (100). The knockout of LCytb/CYB561A3 can cause catastrophic damage to lysosomes and mitochondria and damage mitochondrial respiratory function (101). A study evaluated the relationship between LCytb/CYB561A3 and tumors, including Burkitt's lymphoma caused by EBV infection (101). CYB561A3 was demonstrated to be critical for the proliferation of Burkitt's lymphoma cells, but to have no effect on the proliferation of lymphoblastic lymphoma cells or other cancer cells subjected to EBV transformation. Although the autophagic degradation of ferritin and plasma membrane transferrin were upregulated, CYB561A3 knockout induced severe iron starvation in Burkitt's cells (101).

SDR2, encoded by the gene FRRS1, is a homolog of CYB561 and Dcytb, which is localized in the cell membrane and expressed at high levels in the liver (62), kidney (62), esophageal mucosa, oral epithelium, gallbladder, testicles and heart, as well as in lesions associated with asthma, dermatitis, and rhinitis (102). A recent study found that FRRS1 is upregulated in cervical squamous cell carcinoma (103). SDR2 exhibits iron chelate reductase activity and functions as an active iron reductase that regulates catecholamines in the brain. It reduces Fe³⁺ to Fe²⁺ prior to transportation from the endosome to the cytoplasm (67,104) (Fig. 3).

FRRS1 has been predicted by machine learning methods to be upregulated in the brain tissue of patients with Alzheimer's disease and suggested to be a potential risk gene for Alzheimer's disease (105). Linton et al (106) found that the expression of FRRS1 is downregulated in primary soft tissue sarcoma of the extremities and is negatively correlated with metastatic recurrence. In addition, they tested the combined prognostic effects of FRRS1, helicase 4, complement Factor H and mesenchymal-epithelial transition factor using a simple equal-weight scoring system, and found that they had a greater prognostic effect than tumor grading. However, by contrast, other researchers found that FRRS1 is upregulated in SiHa and HeLa cervical squamous cell carcinoma cells, and transfection with short hairpin-FRRS1 inhibited the growth of these cells and promoted their apoptosis (103).

Encoded by CYB561D2, 101F6 is highly expressed in the pancreas, nervous system, granulocytes and human glioma (107). It is a transmembrane reductase with iron reductase activity (108), which uses ASC in the cytoplasm as an electron donor to transfer electrons through the endoplasmic reticulum for reduction of lumenal MDA and Fe³⁺ (109–111) (Fig. 3).

CYB561D2 is a putative tumor suppressor gene that is located in the 3p21.3 region of the human chromosome, where allelic deletions and genomic changes are frequently found in lung cancer and numerous other cancers (112–114). The overlap of heterozygosity deletion and homozygosity deletion in this region occurs frequently in lung and breast cancer, suggesting that one or more genes in this region play an important role in the pathogenesis of these cancers (112–115). Recombinant adenovirus-mediated transfection of 101F6 demonstrated that 101F6 inhibits cell growth alters the cell cycle and induces apoptosis in human lung cancer cells (116). In addition, the exogenous expression of 101F6 enhances the uptake of ASC by lung cancer cells, leading to the accumulation of cytotoxic H₂O₂. This cooperatively kills tumor cells through apoptosis and autophagy pathways, independently of caspase activation (116). By contrast, the upregulated expression of CYB561D2 has been found to be associated with a higher clinical grade and shorter survival time in patients with glioma. In vitro experiments revealed that the overexpression of CYB561D2 in glioma increased the expression of the immunosuppressive genes PD-L1, chemokine (C-C motif) ligand 2 and tryptophan 2,3-dioxygenase in co-cultured T cells (117), while CYB561D2 knockout inhibited the growth, colony formation and migration of glioma cells and promoted cell apoptosis (117).