**2. Profilin family**

#### **2.1 Gene expression, products & intracellular localization of profilins**

So far, there are four profilin genes that have been identified in the mouse and humans. Normally, the isoforms are expressed by diverse genes; nevertheless, differentially spliced isoforms are known to be present as well. It has been reported that in human, bovine, mouse, and rat, profilin-II is alternatively spliced into profilin-IIA and -IIB (Di Nardo et al., 2000; Lambrechts et al., 2000). In humans, profilin-I is expressed in every cell, while other isoforms are expressed in specific tissues. For example, profilin-IIA and -IIB are found to be brain specific and they are essential for neuronal development (Witke et al., 2001). Profilin-II complexes with other proteins such as synapsin and dynamin-I, well- known proteins that implicated in membrane trafficking. In addition, in humans and mouse profilin-III has been shown to be expressed in the testis and kidney and entirely in developing spermatids (Braun et al., 2002). At the amino acid level profilin-III and -IV exhibited only 30% identity among themselves and with other mammalian profilins (Obermann et al., 2005). Profilin-IV plays a key role in acrosome production and sperm morphogenesis. The same study by Obermann et al. proposes that profilin-III and -IV are transcribed in the germ cells. Yet, the expression timing was different during the rat testis post-natal development and in the rat spermatogenetic cycle. In the human testis, there is a correlation between profilin-IV mRNA expression and the presence of germ cells. Profilin-III and -IV may control testicular actin cytoskeleton dynamics and be a factor in acrosome production and spermatid nuclear shaping (Obermann et al. 2005).

Additionally, in *Caenorhabditis elegans* three profilin isoforms, profilin-I, profilin-II, and profilin-III, have been reported, among them profilin-I is crucial; however, profilin-II and profilin-III are not (Polet et al., 2006). As evident by immunostaining expression patterns for the profilin isoforms was different. At the early stages of embryogenesis, profilin-I confines to the cytoplasm and to the cellular contacts, while at the later stages of embryogenesis it confines to the nerve ring. At the late stages of embryogenesis, it has been shown that profilin-III expresses exclusively in the muscle cell walls. On the other hand, during adulthood, profilin-I is expressed in the neurons, the vulva, and the somatic gonad, profilin-II in the intestinal wall, the spermatheca, and the pharynx, and profilin-III, as dots, in the muscle cells of the body wall (Polet et al., 2006). Furthermore, two profilin isoforms (I and II) have been identified in *Dictyostelium amoebae*; profilin-I is fundamental

Zuo et al., 2007). Another clinical problem in which profilins may be involved is the lateral spreading of some infectious diseases (Pistor et al., 1995; Smith et al., 1996; Zeile et al., 1996). Moreover, profilins got a clinical consideration in other unexpected milieu. In this regard, profilins have been reported as major allergens implicated in pollen and food allergies in approximately 20% of type I allergy patients (Ebner et al., 1995; Valenta et al., 1991c, 1992). Furthermore, we (Hassona et al., 2010, 2011; Moustafa-Bayoumi et al., 2007) and others (Caglayan et al., 2010; Romeo & Kazlauskas, 2008; Romeo et al., 2004, 2007) have shown that profilin-I is an unexpectedly novel molecule that plays a highly significant role in vascular problems that predict a higher risk for developing arteriosclerosis, hypertension, stroke, heart failure, and finally death. Therefore, the aim of this chapter is to shed light on the significance of profilin-I via understanding the molecular and cellular aspects of this

So far, there are four profilin genes that have been identified in the mouse and humans. Normally, the isoforms are expressed by diverse genes; nevertheless, differentially spliced isoforms are known to be present as well. It has been reported that in human, bovine, mouse, and rat, profilin-II is alternatively spliced into profilin-IIA and -IIB (Di Nardo et al., 2000; Lambrechts et al., 2000). In humans, profilin-I is expressed in every cell, while other isoforms are expressed in specific tissues. For example, profilin-IIA and -IIB are found to be brain specific and they are essential for neuronal development (Witke et al., 2001). Profilin-II complexes with other proteins such as synapsin and dynamin-I, well- known proteins that implicated in membrane trafficking. In addition, in humans and mouse profilin-III has been shown to be expressed in the testis and kidney and entirely in developing spermatids (Braun et al., 2002). At the amino acid level profilin-III and -IV exhibited only 30% identity among themselves and with other mammalian profilins (Obermann et al., 2005). Profilin-IV plays a key role in acrosome production and sperm morphogenesis. The same study by Obermann et al. proposes that profilin-III and -IV are transcribed in the germ cells. Yet, the expression timing was different during the rat testis post-natal development and in the rat spermatogenetic cycle. In the human testis, there is a correlation between profilin-IV mRNA expression and the presence of germ cells. Profilin-III and -IV may control testicular actin cytoskeleton dynamics and be a factor in acrosome production and spermatid nuclear

Additionally, in *Caenorhabditis elegans* three profilin isoforms, profilin-I, profilin-II, and profilin-III, have been reported, among them profilin-I is crucial; however, profilin-II and profilin-III are not (Polet et al., 2006). As evident by immunostaining expression patterns for the profilin isoforms was different. At the early stages of embryogenesis, profilin-I confines to the cytoplasm and to the cellular contacts, while at the later stages of embryogenesis it confines to the nerve ring. At the late stages of embryogenesis, it has been shown that profilin-III expresses exclusively in the muscle cell walls. On the other hand, during adulthood, profilin-I is expressed in the neurons, the vulva, and the somatic gonad, profilin-II in the intestinal wall, the spermatheca, and the pharynx, and profilin-III, as dots, in the muscle cells of the body wall (Polet et al., 2006). Furthermore, two profilin isoforms (I and II) have been identified in *Dictyostelium amoebae*; profilin-I is fundamental

molecule, and its role in the vascular diseases.

**2.1 Gene expression, products & intracellular localization of profilins** 

**2. Profilin family** 

shaping (Obermann et al. 2005).

for growth and development, where profilin-II is not. Moreover, it has been reported that *Saccharomyces cerevisiae* and *S. pombe* have only a single profilin isoform (Ezezika et al., 2009; Magdolen et al., 1988).

Based on the small sizes of profilin (15 kDa) and the profilactin complex (57 kDa) one might expect that they can easily diffuse to the nucleus. Nonetheless, profilin ordinarily is excluded from the nucleus and can be found only in the cytoplasm. Either the most part of profilin is bound in the cytoplasm and only a small portion can diffuse freely or there is a particular export mechanism that can actively take the profilin out of the nucleus (Witke, 2004). Recently, Stuven et al., (2003) reported a profilin-specific exportin present in the mammalian cells. Exportin 6 identifies the actin-bound profilin only, as a cargo and moves it out of the nucleus. The reasons for the existence of this profilactin-specific exportin still unclear, but this finding proposes that the nuclear levels of profilin and actin should be strictly regulated (Witke, 2004). Conversely, there are numerous reports about a nuclear fraction of profilin. For example, it has been reported that profilin-I is linked with subnuclear structures such as ribonuclear particles and Cajal bodies, and anti-profilin antibodies interfere with splicing *in vitro*. This implies a role for profilin-I in pre-mRNA processing (Skare et al., 2003). Also, it has been proposed that in the nucleus profilin-I and profilin-II interact with the survival of motor neuron (SMN) protein, a nuclear factor that is mutated in spinal muscular atrophy (Giesemann et al., 1999). SMN is important for splicing regulation yet it is not known whether this requires profilin binding or not. Still, in cell culture, co-localization of profilin-I and profilin-II with SMN in nuclear gems has been established (Giesemann et al., 1999).

To date the nuclear localization of profilins is a mystifying finding. Only a role for profilin and actin in splicing, chromatin remodeling or transcriptional regulation can be speculated. A more detailed understanding of the dynamics and properties of nuclear profilin and actin is required. It is possible that in the nucleus these proteins are considered necessary momentarily during the cell cycle or, particularly, in cells experiencing transcriptional activity changes (Witke, 2004).

#### **2.2 Structural aspects of profilin**

All recognized profilins share common structural and biochemical properties, though the amino acid sequences of the analogous isoforms in distantly related species may demonstrate less than 25% homology (Schlüter et al., 1997). Numerous studies on profilins from different origins demonstrate that they have highly similar tertiary structures (Fedorov et al., 1994, 1997; Metzler et al., 1993; Schutt et al., 1993; Thorn et al., 1997; Vinson et al., 1993) (Figure. 1). The profilin polypeptide consists of 100-131amino acids (Krishnan & Moens, 2009) and it is folded into a central β-pleated sheet formed of 5–7 antiparallel β-strands (Schlüter et al., 1997). On one side, this core is flanked by N- and C-terminal α-helices, with both termini next to each other, and on the opposed side by an extra α -helix attached to either additional α -helix or a small β-strand (Schlüter et al., 1997) (Figure. 1).

It has been reported that there are three groups of ligands characterize profilins: (1) G-actin and actin-related proteins (Machesky et al., 1994; Schutt et al., 1989; Tobacman et al., 1983) (2) polyphosphoinositides (Lassing & Lindberg, 1985, 1988) (3) poly-L-proline (PLP) with the exception of *Vaccinia* profilin (Kaiser et al., 1989; Lindberg et al., 1988; Tanaka & Shibata 1985), existing either as a peptide or as a sequence motif in particular proteins.

In this context, Gieselmann et al., (1995) showed that human profilin-I exhibits about five folds higher affinity for actin than profilin-II. Radiography analyses of the structures of human profilin isoforms imply that the substitution of profilin-I S29 by Y29 in profilin-II participates in the higher affinity of profilin-II for proline-rich sequences (Nodelman et al., 1999). In spite of the similarity in the 3D structures of human profilin-I and -II, the surface characteristics, such as exposure of hydrophobic patches (Figure 2), and biochemical properties of each isoform are different (Krishnan & Moens, 2009).

Fig. 1. Profilin-I isoforms from different organisms showing a similar helix (red) and strand (cyan) structure (PDB database: 1PFL, 1KOK, 2PRF, and 3NUL) with the loops highlighted in green, adapted from Krishnan & Moens, (2009) with permission.
