**1. Introduction**

Transcription factors are a group of proteins that exhibit the ability to bind to genetic material (DNA). The site of attachment of transcription factors in the DNA region may be a promoter or an enhancer sequence in a specific site or region that regulates the transcription process. The action of transcription factors can be selectively activated in the cell or deactivated by other cellular proteins which usually takes place at the last stage of signal transmission in the cell [1]. One of the more important families among transcription factors is the FOX family, which plays an important role in regulating the expression of genes involved in cell growth, proliferation, and differentiation. This family includes the Foxp3 protein (Forkhead box protein P3) which is not only a transcription factor but also a key molecule involved in the development of regulatory T cells (Treg) [2,3]. It plays an important role in maintaining the homeostasis of the immune system, enables the complete stability of the Treg lineage, and directly modulates the expansion and function of conventional T-cells [3,4]. The Foxp3 protein can act both as a repressor and activator of the transcription process, while the type of its activity depends on its interaction with other transcription factors present in the cell [4]. Disturbances in the proper functioning of the Foxp3 protein in the human body (usually caused by mutations within the coding

**Citation:** Mertowska, P.; Mertowski, S.; Podgajna, M.; Grywalska, E. The Importance of the Transcription Factor Foxp3 in the Development of Primary Immunodeficiencies. *J. Clin. Med.* **2022**, *11*, 947. https://doi.org/ 10.3390/jcm11040947

Academic Editors: Andreas Hecker, Rita Consolini and Giorgio Costagliola

Received: 31 December 2021 Accepted: 9 February 2022 Published: 11 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sequences) dysregulate the immune homeostasis and leads to the development of diseases called immunodeficiencies [5,6]. This is a special group of disorders in which the patient's immune system is characterized by a reduced ability or lack of defense against various pathogens. It is manifested by frequent, recurrent infections with microorganisms (bacteria, viruses, fungi) that are resistant to long-term therapy [7]. In the vast majority of cases it is caused by microbes commonly inhabiting the human body which, as a result of weakened immune reactions, cause the so-called opportunistic infections [8]. Apart from the tendency of recurrent infections, immunodeficiency is also accompanied by other health problems such as allergic phenomena [9], autoimmune phenomena [10,11], granulomas [12], tumors [13,14], endocrine disorders [15,16], and various cytopenias (most often thrombocytopenia [17] and neutropenia [18,19]) as well as diseases of the lungs [20] and gastrointestinal tract [21] (e.g., irritable bowel syndrome) [22,23]. Due to the underlying causes of immunodeficiencies, we can divide them into two groups: Primary Immunodeficiencies (PID), which are genetically determined and associated with mutations that are either inherited or appear de novo and Secondary Immunodeficiencies (SID), which are caused by external factors or the presence of comorbid disease [24,25].

The aim of this review is to present the role of the Foxp3 transcription factor in the human body in maintaining immune homeostasis and its significance during the development of immunodeficiency. Due to the Foxp3 protein's involvement in the regulation of transcription, we will try to explain its role in the development of primary immunodeficiency diseases that are strongly associated with genetic disorders.

#### **2. Molecular Characterization of the Foxp3 Protein**

The human *FOXP3* gene is located on the X chromosome, more specifically, by genomic sequence analysis, it was shown to be on the p arm at position Xp11.23 [26,27]. The gene is composed of 12 exons whose exon-intron boundaries are identical in the gene coding regions of both mice and humans [28]. The degree of nucleotide sequence identity between these organisms is 84.98% [26–28]. The *FOXP3* gene is responsible for the encoding of a protein product composed of 431 amino acids with a total molecular weight of 47.27 kDa and an isoelectric puncture of 8.62 (Table 1). The analysis of the protein sequence in the Foxp3 protein showed that its structure contains 47.33% hydrophobic amino acids and 52.67% hydrophilic amino acids (Table 1). According to the literature, the Foxp3 protein can also occur in the human body in the form of three other isoforms: isoforms 2, 3, and 4 [29]. They differ mainly in amino acid length and molecular weight which changes the amino acid composition, isoelectric point, and secondary structure of the protein. Our research team performed a detailed analysis of the similarity of the amino acid sequence of the Foxp3 protein and its three isoforms using the UniProt database. The differences made on this basis are shown in Table 1 and Figure 1.

**Table 1.** Characteristics of the basic properties of the Foxp3 protein and its isoforms.


**Figure 1.** Characterization of the amino acid sequence of the Foxp3 protein and its isoforms (**A**) Comparison of the amino acid sequence of the Foxp3 protein and its isoforms; (**B**–**E**) Amino acid scald of Foxp3 protein and its isoforms [own elaboration]. Marks: (\*) means the amino acids are identical in the sequence, while (-) means the lack of amino acids in the sequence.

For isoform 2, 34 amino acids are missing from the Foxp3 protein sequence (located at positions 72–106). The same is the case for isoform 3 where at position 382, a fragment consisting of 61 amino acids (-KVSSSEVAVTGMASSAIAAQSGQAWVWAHRHIGEERDVGCW WWLLASEVDAHLLPVPGLPQ-) is also added [29]. Additionally, between the Foxp3 protein and isoform 4, there is a difference of 26 amino acids located between amino acids 246–272 which determines the highest degree of sequence identity. The degree of identity of the amino acid sequence of the Foxp3 protein and its isoforms is quite diverse and ranges from 75.15% (between isoforms 3 and 4) to 93.73% (between the Foxp3 protein and isoform 4). Detailed information on the degree of amino acid sequence identity between individual proteins is provided in Table 2.


**Table 2.** The degree of identity of the amino acid sequence of the Foxp3 protein and its isoforms.

Foxp3 isoforms differ not only in their structure and the degree of similarity of the amino acid sequence but also in their ability to interact with different proteins. The Foxp3 protein is able to interact with the IKZE3 protein (via the LXXLL motif), while as reported in the literature, isoform 2 does not [4,30]. It has been shown that it is able to interact with the ZFP90 protein and create complexes with the TRIM28 protein [31]. However, further research is needed to understand the differential function of Foxp3 isoforms.

The Foxp3 protein has several distinctive motifs in its structure. Two of them, located between amino acids 68–76 and amino acids 239–248, are nuclear export signals which are short peptides containing hydrophobic residues targeted for export from the cell nucleus into the cytoplasm through the nuclear pore complex (Figure 2) [32]. Another example is the LXXLL motif, located between amino acids 92–96, which is involved in many proteinprotein interactions related to various aspects of transcription regulation (Figure 2) [33–35]. These motifs are present in many transcription factors and cofactors, mediating interactions that may activate or suppress transcription [36,37]. Several recently reported 3D structures of protein-LXXLL motif complexes have been associated with leukemia, further highlighting the diversity and regulatory importance of this seemingly simple motif [36,38,39].

**Figure 2.** Motifs and domains occurring in the amino acid sequence of the Foxp3 protein (own elaboration based on the Uniprot database [29]).

Another motif located between amino acids 414–417 is the nuclear localization signal which is an amino acid sequence that "tags" a protein for import into the cell nucleus via nuclear transport (Figure 2). Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface [40]. Two extremely important domains within the sequence of this protein should also be noted. The first is a zinc finger domain of 26 amino acids located in the region between amino acids 197 and 222 (Figure 2). It is a structure found in DNA binding proteins and is directly involved in the binding of a nucleic acid molecule by a protein [41]. The presence of the zinc ion (Zn2+) in the domain is crucial for the stability of the entire structure as its absence may result in structural and functional changes. The zinc atom in this structure is coordinated by residues C198, C203, H216, H221, and partially by D220 [42,43]. Near the zinc finger domain, there is another leucine zipper motif (239–260 amino acids) which is very important due to the functions performed by the Foxp3 protein [44,45]. This motif mediates intermolecular interactions, indicating the possible interaction in the dimerization process which is the essential function of the transcriptional regulator [45]. As indicated by research data, this motif is involved not only in homo-association but also in hetero-association with the Foxp1 protein [44,45]. Additionally, it has been shown that the presence of mutations within the leucine zipper motif can significantly reduce the binding affinity of the Foxp3 protein to the promoter regions in vitro [42]. The discoveries made by Mackey-Cushman et al., (2011) showed that the leucine zipper motif also mediates interactions between the Foxp3 protein and histones (the H1.5 histone, precisely) which suppress interleukin 2 (IL-2) transcription in T cells [46]. It should also be mentioned that the zinc finger motif is not directly involved in the dimerization process and the very mechanism of this process is not fully understood [42]. The second extremely important structure is the domain called "fork-head", which is often described as the transcription factor, whose purpose is to bind DNA. In the case of the Foxp3 protein, this domain is 87 amino acids long and is located between amino acids 337–423 in the sequence (Figure 2) [47,48]. It is involved in the protein dimerization process and its interaction with nuclear factor NFATC2 is responsible for activating T cells [49]. The NFATC2 protein is present in the cytosol and translocates only to the nucleus upon stimulation of the T-cell receptor (TCR) where it becomes a member of the nuclear factors of the activated T-cell transcription complex. This complex plays a key role in inducing gene transcription during the immune response [49,50].
