In the nucleus, the structural protein nonerythroid α spectrin (αSpII) plays a central role in repair of DNA interstrand crosslinks (ICLs). Significantly, there is a deficiency in αSpII in the genetic, bone marrow failure disorder, Fanconi anemia (FA), which is characterized by a defect in DNA ICL repair. This is due to excessive cleavage of αSpII by the protease, µ-calpain. Importantly, knockdown of µ-calpain, by siRNA, reverses cleavage of αSpII, restores its levels to normal and enables repair of DNA ICLs in FA cells. FA thus represents another disorder in which excessive cleavage of αSpII by µ-calpain correlates with a major characteristic, in this case defective DNA ICL repair. It demonstrates the importance of αSpII for the DNA ICL repair process. FA proteins are proposed to play a major role in regulating cleavage of αSpII by µ-calpain thus giving them another critical role in DNA repair.

Cytoskeletal and nucleoskeletal proteins are critical for maintaining the structure, function and mechanical properties of eukaryotic cells [1–11]. Of particular importance is spectrin, found in both erythroid and nonerythroid cells [12–19]. Spectrin was first identified in erythrocytes and shown to be essential for cell membrane structure, integrity, and flexibility [14, 15, 18, 20–24]. Spectrin also plays a critical role in non-erythroid cells in both the cytoplasm and nucleus and is involved in maintaining the shape, flexibility and elasticity of both the cell and nuclear membranes and is essential for a number of functionally important cytoplasmic and nuclear processes, as well as for mechanical coupling between the nucleus and the cytoplasm [7, 10, 13–17, 25–39]. Total loss of spectrin can lead to cell death [33, 36, 38, 40]. Nonerythroid spectrin has two subunits, αSpII and βSpII [14–17, 34, 35]. Of considerable interest, nuclear αSpII has been shown to directly interact with DNA and play a critical role in DNA repair and in the maintenance of the stability of chromosomes and telomeres after DNA damage [12, 13, 38, 41–46]. It acts as a scaffold and aids in recruitment of repair proteins to sites of damage in both telomeric and non-telomeric DNA [42–49].

Loss or breakdown of spectrin in cells leads to a deficiency in these processes and is clinically manifested in a number of disorders, one of which is the genetic disorder, Fanconi anemia (FA) [13, 38, 44, 48, 50]. FA is characterized by bone marrow failure, a marked propensity to develop cancer, multisystemic congenital abnormalities, chromosomal instability and a defect in ability to repair DNA interstrand cross-links (ICLs) [51–69]. Lambert et. al. have shown a deficiency in αSpII in cells from FA patients from a number of FA complementation groups [12, 13, 44, 48]. This deficiency is due to the excessive activity of the protease, µ-calpain, which cleaves αSpII leading to its breakdown [69, 70]. The breakdown of αSpII correlates with a defect in DNA ICL repair in FA cells in both nontelomeric and telomeric DNA and to chromosome instability [46, 69]. These studies have demonstrated that maintaining the stability of αSpII in the cell is critical for a number of important nuclear and cellular processes and for circumventing telomere dysfunction after DNA ICL damage [46, 69]. Lambert et al. have proposed that excessive cleavage of αSpII by μ-calpain is an important factor in the pathogenesis of FA and a number of the clinical characteristics of this disorder [13, 50, 69].

This review will concentrate on the function of αSpII in the nucleus with emphasis on its interaction with non-telomeric and telomeric DNA, especially after DNA ICL damage, the importance of maintaining the stability of αSpII and preventing its breakdown by μ-calpain, and the role of FA proteins in this process. The consequences of a loss or deficiency in αSpII on DNA repair, telomere integrity/function and chromosome stability after DNA damage, particularly in FA, will be discussed.

αSpII is a long flexible protein which contains a modular structure composed of an extended array of triple α-helical repeats connected by short a-helical linker [14–17, 21, 27, 71–73]. This structure aids in its flexibility and ability to expand and contract [14, 25, 71–73]. Nonerythroid spectrin has two subunits, αSpII and βSpII. There is one alpha spectrin gene and four beta spectrin genes [14–17]. There are a number of different spectrin isoforms which originate by extensive mRNA splicing from these genes [14, 27]. In all of these, αSpII and βSpII associate in an antiparallel fashion to form a heterodimer [14–17]. αSpII consists of 21 triple helical repeats and βSpII contains 17 repeats [14–17]. Two heterodimers can link head to head to form a tetramer [14–17]. Nonerythroid spectrin (αSpII/βSpII) is comprised of numerous domains. αSpII contains an EF hand’s domain on repeat 21, which is a site of Ca²⁺ binding and signaling (Figure 1) [14–17]. It contains a Src-homology 3 (SH3) domain in repeat 10, which plays an important role in proteinprotein interactions and is involved in signal transduction and intracellular signaling (Figure 1) [14–17, 74, 78–81]. The SH3 domain has a binding site for a kinase, c-Src, lowmolecular weight phosphotyrosine phosphatase (LMW-PTP) and FANCG, a Fanconi anemia (FA) protein. αSpII has a site of cleavage by the protease, calpain, in repeat 11 between residues Y₁₁₇₆ and G_1177. [75, 76] There is a caspase cleavage site at residue Asp1185, which is activated during apoptosis [82]. αSpII also has a calmodulin binding domain in repeat 11, which can modulate spectrin cleavage by µ-calpain and caspase [83, 84]. Proteolytic cleavage of αSpII by µ-calpain leads to its decreased stability and loss of function, which makes regulation of µ-calpain cleavage of αSpII critical for maintaining normal cell and nuclear functioning [74, 76, 78]. There is also a ubiquitination site in repeat 21 and a suggested E2/E3-ubiquitin-conjugating/ligating site in repeat 20 [14, 26]. βSpII has an actin-binding domain on repeat 1, and a pleckstrin homology domain on repeat 7, which is involved in cell signaling, organization of the cytoskeleton, and aiding in the localization of βSpII to the plasma membrane [14–17]. αSpII and βSpII assemble laterally to form heterodimers that assemble into tetramers [14–17]. The tertiary structure of the spectrin repeat imparts elasticity to the protein [14–17, 21, 27, 71–73]. The structural characteristics of αSpII/βSpII enable it to interact with a number of different cellular proteins and participate in a number of important physiological pathways in the cell in both the nucleus and the cytoplasm. Numerous reviews are available with a moredetailed description of spectrin structure [14–17, 20, 24–27, 71–73].

Spectrin is a multifunctional protein, as is evidenced by the numerous proteins which interact with both αSpII and βSpII. Goodman and colleagues have created the Spectrinome using Human Proteome, Human Reactome and Human Atlas data which clearly demonstrates the multitude of cellular pathways and functions in which spectrin is involved in the cytoplasm, the nucleus and the cell surface [14]. Since spectrin’s numerous interacting protein partners demonstrate the multitude of roles spectrin plays in the cell, it is important to more fully understand these interactions and their impact on cellular function.

One of the best known and critical roles of αSpII/βSpII is providing structural integrity for cell membranes [14, 19, 85, 86]. It acts as a scaffold interacting with ankyrin, actin and other cytoskeletal proteins to form a network crucial for maintaining mechanical support, shape, flexibility and elasticity to the cell membrane [17, 24, 85–89]. αSpII is involved in cell movement and adhesion via interactions with proteins involved in actin dynamics (Figure 2) [31–33, 89]. αSpII plays a role in actin filament reorganization which is critical for a number of processes such as lamellipodia extension and immunological synapse formation between T-cells and antigen-presenting cells [27, 32, 33, 89]. The SH3 domain of αSpII has been shown to bind to proteins involved in actin polymerization and reorganization and plays a crucial role in this process during cell-cell contact, cell adhesion, cell spreading and migration [29, 33, 89–91]. Spectrin interacts with proteins involved in intracellular traffic. It is involved in vesicle and organelle mediated transport [14, 92]. In neurons, αSpII/βSpII and αSpII/βSpIII have an essential role in synaptic vesicle trafficking and synaptic transmission [14, 16, 93]. βSpIII associates with the microtubule motor proteins, kinesin and dynein, and is involved in anterograde and retrograde transport of cargo in axons [14, 94–96]. αSpII and βSpII associate with synaptic vesicles via synapsin I and are critical for this process [14, 28, 29, 97]. αSpII and βSpII have been identified associated with melanosomes in human melanocytes and are thought to be involved in melanosome transport in these cells [98]. Goodman et al have also shown that erythroid spectrin has E2/E3 chimeric ubiquitin conjugating and ligating activity and is capable of ubiquitinating not only alpha spectrin but also ankyrin, band 3, and protein 4.1 [14, 26, 99, 100]. Goodman et al. have proposed that αSpII has similar E2/E3 ubiquitinating activity in the cytoplasm and the nucleus in non-erythroid cells [14, 26, 99, 100]. This activity is very important for protein-protein interactions and the role they play in normal physiological processes in the cell. Thus, αSpII along with βSpII are components of a spectrin scaffold which is critical for a large number of physiological processes in the cytoplasm.

Just as interactions of spectrin with specific proteins in the cytoplasm are critical for cellular function, its interactions with both proteins and DNA in the nucleus is critical for nuclear function. Within the nucleus, nonerythroid spectrin is a component of a nucleoskeletal network which plays an essential role in maintaining the structural integrity and elasticity of the nucleus as well as in repair of damage to both nontelomeric and telomeric DNA (Figure 2) [7, 10, 15–17, 27, 37, 46, 50, 101]. Both αSpII and two isoforms of β-spectrin (βSpII and βSpIV∑5) have been identified in the nucleus [12, 13, 38, 39]. Studies have shown that αSpII is present in the peripheral nucleoskeleton, where it interacts with lamins A and B, actin, and nuclear myosin and is anchored to the nuclear envelope by emerin and protein 4.1 [7, 8, 10, 13, 37, 41, 50, 102]. It makes an important contribution to the biomechanical properties of these nucleoskeleton proteins and to nuclear envelope support [7, 10, 37, 102]. It forms a complex with lamins A and B, actin, nuclear myosin, and the LINC complex protein, SUN2, and plays an important role in coupling the nucleoskeleton to the cytoskeleton [7, 10, 13, 37, 41]. αSpII is also associated with the inner nucleoskeleton where it interacts with DNA repair proteins, chromatin remodeling proteins and proteins involved in transcription and RNA processing [6–8, 13, 41, 42].

The cortical network αSpII forms with lamin and actin at the nuclear envelop plays a role in maintaining the elasticity and structural integrity of the nucleus (Figure 2) [7, 10, 15, 17, 27, 37]. Knock-down of αSpII in HeLa cells to levels that are approximately 50% of those found in control cells leads to a decrease in ability of the nucleus to recover from compression [37]. This has been attributed to loss of membrane elasticity due to decreased αSpII [37]. αSpII’s modular structure of triple alpha-helical repeats makes an important contribution to the structural stability, elasticity and mechanical resilience of the nucleus and to its ability to recover from compression [37].

Thus, αSpII is an important scaffolding protein which represents 2–3% of the total protein in eukaryotic cells [14]. It has a myriad of functions and any defects or deficiencies in this protein would be central to pathological changes occurring in the cell structure and function (Figure 2). Therefore, maintaining αSpII stability is critical for normal cell functioning. Excessive cleavage of αSpII can upset normal cellular homeostasis and result in various human disease states and disorders. These points will be discussed below.

In addition to its structural role in the nucleus, αSpII has been shown to have an essential function in the nucleus in repair of DNA damage. Lambert and colleagues have demonstrated that in human cells αSpII plays a critical role in repair of DNA interstrand cross-links (ICLs) in both non-telomeric and telomeric DNA and is needed for chromosomal stability after DNA damage [13, 42–49, 69]. There are numerous lines of evidence which demonstrate the important role of αSpII in DNA repair: (1) αSpII binds directly to DNA at sites of ICLs; (2) αSpII recruits repair proteins to these sites; (3) αSpII is needed for production of incisions produced by the endonuclease XPF-ERCC1 at sites of ICLs; and (4) αSpII is critical for repair of telomeric DNA and telomere integrity after DNA ICL damage [42–46, 69]. Thus, αSpII is important in DNA repair processes in the nucleus and maintenance of telomere integrity after DNA damage. These points will be discussed below.

The importance of αSpII stability in DNA repair is seen in cells from patients with Fanconi anemia (FA) which, as mentioned above, are defective in ability to repair DNA ICLs [43–48, 51–61]. Of significance, Lambert et. al. have shown that there is a deficiency in αSpII in cells from patients from a number of FA complementation groups [12, 13, 45, 69]. Levels of αSpII are 35–40% of those found in normal human cells [12, 13, 45]. This deficiency correlates with reduced levels of repair of DNA ICLs in FA cells which are 35–45% of normal [45, 47, 48]. Levels of DNA repair after DNA ICL damage were determined in FA-A, FA-C, FA-D2, FA-F and FA-G cells by measurement of unscheduled DNA synthesis (UDS), which measures uptake of nucleotides into DNA repair patches [47].

An important question is, what is the cause of the reduced levels of αSpII in FA cells? Lambert et al have shown that this is not due to reduced expression of αSpII, since FA cells express normal levels of αSpII mRNA [70]. They have demonstrated that it is due to increased cleavage of αSpII as a result of excessive activity of the protease, μcalpain, which cleaves αSpII [69]. They have proposed that FA proteins play a critical role in regulation of μ-calpain cleavage of αSpII and that reduced levels of FA proteins in FA cells leads to increased μ-calpain activity and increased cleavage of αSpII [50, 69]. Thus, both increased μ-calpain activity and reduced levels of FA proteins are proposed to play a critical role in the breakdown of αSpII that occurs in FA cells and the reduction in DNA repair. These points and their significance will be discussed below.

FA serves as an excellent model for the effects that a deficiency in αSpII has on telomere function after DNA damage. As mentioned above, levels of αSpII in FA-A cells are approximately 35–40% of normal [12, 45, 48]. Telomere dysfunction has been observed in FA complementation group A (FA-A) cells after DNA ICL (i.e., MMC or psoralen plus UVA light) damage [46]. This includes a significant increase in TIF formation and a catastrophic loss of telomeres (Figure 10) [46]. This increase correlates with loss of αSpII in these cells, which results in a significant decrease in recruitment of XPF to sites of damage at telomeres as mentioned above (Figure 9A) [46]. These studies indicate that there is a defect in repair of ICLs at telomeres in FA-A cells similar to that seen in normal cells after αSpII has been knocked down.

Evidence that loss of αSpII in FA-A cells is a significant factor in the increase in telomere dysfunction and in the catastrophic loss of telomeres that occurs after DNA ICL damage is demonstrated by studies in which μ-calpain has been knocked down by siRNA. Knock down of μ-calpain in FA-A cells leads to restoration of αSpII levels to those found in normal cells and to reduction in the number of TIF positive cells after MMC treatment [46]. In addition, it also results in a significant decrease in the frequency of signal-free ends (SFEs) or telomere loss in FA-A cells after DNA ICL damage (Figure 10) [46]. These studies thus demonstrate that breakdown of αSpII in FA-A cells due to excessive μ-calpain activity results in telomere dysfunction and loss of telomeres, which can be corrected by knocking-down μ-calpain.

In FA-A cells, unlike in normal cells, XPF does not localize to telomeres after DNA ICL damage (Figure 9B) [46]. However, when μ-calpain is knocked down by siRNA and αSpII levels have been restored to normal, XPF co-localizes with αSpII in damage induced foci at telomeres (Figure 9B) [46]. These studies again demonstrate that αSpII plays a critical role in telomere function after DNA ICL damage, and that restoration of αSpII to normal levels in FA cells by knocking down μ-calpain can have a significant effect on increasing the DNA repair capability of these cells and correcting the telomere dysfunction observed after DNA ICL damage [46].

Chromosomal aberrations produced after DNA ICL damage are also corrected in FA cells after restoration of αSpII levels to normal by knocking-down μ-calpain [45, 46]. The aberrations which were corrected include sister chromatid end-to-end fusions, chromosome exchanges, and chromatid breaks and radials and are similar to those produced after ICL damage in normal cells in which αSpII levels have been knocked down [45, 46]. Thus decreasing μ-calpain activity in FA cells and restoring αSpII levels to normal leads to restoration of DNA repair capabilities, localization of XPF to sites of DNA damage, increased chromosomal stability, and a decrease in formation of dysfunctional telomeres and in loss of telomeres after DNA ICL damage. These studies indicate that both loss of αSpII and telomere dysfunction in FA-A cells may be important factors in the chromosomal aberrations which develop after DNA ICL damage.

Of particular interest, FA-A cells in which αSpII levels have been restored to normal are still deficient in the FANCA protein. The question that arises is how DNA ICL repair can still proceed in the absence of this protein. Lambert et. al. have proposed that FA proteins, in addition to a role in DNA repair, are also important in maintaining the stability of αSpII and reducing its cleavage by μ-calpain [46, 69]. When αSpII stability has been restored in FA-A cells by knocking-down μ-calpain, DNA repair is able to proceed, possibly involving an alternative means [49, 50]. In FA-A cells, in which there is a deficiency in FANCA, FANCD2 is not monoubiquitinated [49]. FANCD2-Ub has been shown to be important for the ICL repair pathway to proceed [57–59]. Lambert et al have shown, however, that in FA-A cells in which αSpII levels have been restored, non-Ub FANCD2 foci are observed at levels approximately 80% of normal just as they are in FA-A cells in which levels of αSpII have not been restored [49]. FANCD2-Ub is not present [49]. The non-Ub FANCD2 foci localize to sites of DNA ICL damage and the nonUb FANCD2 foci follow the same time course for formation of nuclear foci after ICL damage as the FANCD2-Ub foci do. However, in the FA-A cells non-Ub FANCD2 foci only appear after levels of αSpII have been returned to normal [49]. Thus non-Ub FANCD2 requires the presence of αSpII in order to localize to sites of damage. αSpII does not colocalize with these non-Ub FANCD2 foci, just as it does not co-localize with FANCD2- Ub foci in normal cells after DNA ICL damage [49]. Lambert et al have proposed that in FA-A cells when levels of αSpII have been returned to normal, non-Ub FANCD2 localizes to sites of damage and plays a role in the DNA repair process [49]. This is potentially an alternate method for DNA ICL repair. Whether this is mainly a back-up mechanism or is also involved in normal ICL repair is not clear. Other studies support the proposal that non-Ub FANCD2 plays a role in DNA ICL repair, in particular, in the nonhomologous recombination step in the repair process and in replication fork recovery [159–163]. The relationship between αSpII and FANCD2-Ub and non-Ub FANCD2 after DNA damage needs to be further investigated.

As noted above, breakdown and loss of αSpII in FA cells has a significant effect on both DNA repair and chromosome stability after damage with a DNA interstrand crosslinking agent. Lambert et al. have proposed that this loss is an important contributing factor in the pathogenesis of FA [38, 44]. There are a number of defining characteristics associated with FA pathogenesis, the major ones of which include a defect in DNA repair, chromosome instability, cancer predisposition, bone marrow failure and multisystemic congenital abnormalities [47, 51–69]. Experimental evidence has shown that loss of αSpII in FA cells has a significant impact on several of these characteristics. Breakdown of αSpII in FA cells has been shown to be directly involved in the defect in ability of FA cells to repair DNA ICLs [13, 44, 45]. Loss of αSpII results in failure of XPF/ERCC1 to localize to sites of DNA ICLs and create incisions at these sites leading to defective DNA ICL repair in FA cells in both telomeric and non-telomeric DNA [13, 44, 47, 50]. In addition, excessive breakdown of αSpII in FA cells also results in chromosome instability and production of chromosomal aberrations after DNA ICL damage [44, 45]. It has been shown that failure to repair DNA ICLs, particularly in S phase of the cell cycle can directly lead to the production of DNA double-strand breaks and chromosomal aberrations [164]. This is proposed to be a major cause of chromosomal aberrations in FA cells after DNA ICL damage [44, 45, 50]. Restoration of αSpII levels to normal in FA cells, by knocking down μ-calpain, leads to restoration of DNA ICL repair and chromosome stability [45, 47, 50]. These studies thus indicate that αSpII is critical for two important cellular processes, DNA repair and chromosome stability of DNA ICL damage, which are defective in FA cells and which contribute to the pathogenesis of the disorder.

Another phenotypic characteristic of FA is an increased incidence of cancer (e.g., acute myeloid leukemia (AML) and squamous cell carcinoma) and hemolytic anemia [67, 68, 77, 165]. There is evidence that a deficiency in αSpII plays an important role in cancer development [68, 166]. Loss of αSpII has been reported in the bone marrow of patients with acute myeloid leukemia (AML) and it has been proposed that a deficiency in αSpII is a contributing factor in the development of AML [165, 167]. Since FA patients can develop AML, it is possible that loss of αSpII is an important component in this process and may be involved in the leukemogenesis and bone marrow failure which can occur [165, 167]. In a number of B-cell malignant lymphomas, there is a strong correlation between loss of αSpII and development of lymphomagenesis [167]. This has led to the suggestion that a deficiency in αSpII is an important factor in lymphomagenesis [167]. Thus breakdown and loss of αSpII in FA cells could play a role in the development of AML and leukemogenesis observed in FA patients and contribute to FA pathogenesis. This needs to be further examined.

FA patients can also have a number of congenital abnormalities, which include radial ray deformities, absent radi, ear malformations, urogenital anomalies, renal deformities, cardiac defects, and neurological abnormalities [67, 168, 169]. αSpII has been proposed to play a role in many developmental processes [36, 170–172]. In αSpII knockout mice the embryos display cardiac, craniofacial and neural tube abnormalities [170]. Cultured fibroblasts from these mice have impaired growth and spreading [170]. These studies indicate that αSpII is important in cellular morphology and development. Since αSpII is critical for and expressed throughout development in mammalian cells, it can be hypothesized that a deficiency in αSpII in FA patients could indirectly have a significant influence on a number of the developmental abnormalities associated with FA.

αSpII is an essential protein in the cell and an important cellular scaffold which is critical for a diverse number of biological processes, therefore, loss or dysfunction of this protein could be of important clinical significance and play a fundamental role in the etiology of a number of diseases [7, 13–17, 25–39]. Two important causes for a loss or deficiency in αSpII are: (1) enhanced cleavage and break-down of αSpII, or (2) mutations in the gene encoding αSpII, SPTAN1, resulting in expression of a defective αSpII. Some disorders in which defects or a deficiency in αSpII are of clinical significance are presented below.

Loss or a deficiency in αSpII is an important factor in the pathophysiology of a number of neurological disorders [135, 169, 173–179]. Mutations in the SPTAN1 gene are associated with a broad range of neurodevelopmental diseases [169, 174, 175]. One of these is early infantile epileptic encephalopathy, or West Syndrome, which is characterized by progressive brain atrophy, severe neurodevelopmental impairment, mental retardation, difficulty walking, and seizures [169, 174–177]. In this disorder, there is a mutation in the SPTAN1 gene leading to a defect in the binding of αSpII to βSpII and to βSpIV, which are needed for organization and maintenance of the neuronal actin-spectrin cytoskeleton, as well as for axonal and dendritic growth and development and neuronal excitabiliity [174–177].

In a number of other progressive neurodegenerative disorders there is increased breakdown of αSpII [132, 135, 140, 141, 178–181]. It has been shown that this is due to excessive activation of calpain which results in increased cleavage of αSpII. Excessive activation of calpain and αSpII cleavage is a common feature of neurodegenerative diseases and of traumatic encephalopathy and leads to loss of neuronal integrity and the neuropathology of many of these disorders [132, 135, 140, 141, 178, 179]. In Alzheimer’s disease, increased breakdown of αSpII has been demonstrated in numerous cells and tissues and is due to excessive calpain activity but not to changes in levels of calpain [141, 171, 177, 182]. It has been proposed that faulty regulation of the neuronal spectrin skeleton and its breakdown by excessive calpain activity can activate a series of events that leads to the ataxia and neuronal degeneration observed in this and other neurodegenerative disorders [135]. Thus, excessive activation of calpain activity in a number of neurodegenerative diseases results in loss of αSpII [132, 135, 140–142].

Central nervous system and various neurological anomalies are also a clinical characteristic of FA patients. These include: brain and spinal cord anomalies, microcephaly, hydrocephalus, cerebellar defects, and development of medulloblastoma [67, 168, 173, 183–186]. Of considerable interest, FA cells have significantly (3-4 fold) increased levels of breakdown of αSpII due to an excessive increase in µ-calpain activity [13, 50, 69]. FA thus represents another disorder in which loss of αSpII is due to its cleavage by µ-calpain. It can be hypothesized that like some neurodegenerative diseases, excessive activation of μ-calpain activity in FA, leading to significant cleavage of αSpII, may be an important contributing factor in the pathological changes observed in the nervous system and in the pathogenesis of FA. It would be of interest to determine whether there is increased μ-calpain activity and αSpII cleavage in neuronal tissue in FA patients with neurological abnormalities and to speculate that, if a relationship between these two factors were found, this would indicate that breakdown of αSpII is an important factor in progression of these abnormalities.

There is evidence that a deficiency in αSpII plays an important role in development of cancers other than AML. In Lynch Syndrome or hereditary nonpolyposis colorectal cancer, where there is a defect in DNA mismatch repair and a deficiency in the mismatch repair protein, MLH1, there is a significant reduction in levels of αSpII in the tumor cells [187–189]. Since MLH1 directly interacts with αSpII, it has been proposed that loss of MLH1 leads to destabilization of αSpII which, in turn, results in a defect in DNA repair and in the tumor development seen in this disorder [187–189]. These studies, along with those noted above, which suggest that αSpII loss is an important factor in lymphomagenesis, collectively indicate that there is strong evidence that a deficiency in αSpII plays an important role in development of a number of different types of cancer.

In both acquired and neonatal congenital heart failure, there is dysfunction of αSpII that is due to excessive calpain activity which results in increased cleavage of αSpII [190, 191]. This has been demonstrated by the presence of the 150 kDa αSpII breakdown product of μ-calpain in the cardiac cells [190, 191]. αSpII is required for normal cardiac structure, function and development and plays a central role in the formation and regulation of key structural and signaling pathways in the heart [190, 191]. Breakdown of αSpII, which can occur in heart disease, has a significant effect on the cardiomyocyte spectrin network and on regulation of normal cardiac function [36, 190, 191]. Thus, breakdown of αSpII by excessive μ-calpain activity is also an important factor in heart failure.

αSpII dysfunction can therefore lead to a variety of disorders, particularly since it is a multifunctional protein which is critical for a multitude of diverse cellular processes. As mentioned above, there is a relationship between clinical manifestations of all of the disorders described and a loss or deficiency in αSpII, which in turn is due to either enhanced cleavage of αSpII by excessive proteolytic cleavage by μ-calpain or expression of a defective αSpII due to a mutation in the SPTAN1 gene. As a result of the diversity of αSpII function, a deficiency in αSpII can lead either directly or indirectly to dysregulation of a number of different cellular pathways and to pathological changes which are manifested in a variety of different disorders. The number of disorders in which deficiencies in αSpII play a direct or indirect role may still not yet be fully recognized.

αSpII is a critically important and functionally diverse protein which represents 2–3% of all proteins in human cells [14–17]. It forms a network under the plasma membrane and within the cytoplasm ensuring the stability of cell membranes and organelles. It acts as a scaffold interacting with different protein binding partners playing an important role in cell development, cell migration, cell cycle, actin filament organization, intracellular trafficking and signal transduction [24–28]. Within the nucleus, αSpII is part of a nucleoskeletal network which is essential for maintaining the structural integrity of the nuclear envelope and elasticity of the nucleus. It is involved in the biomechanical coupling of the nucleoskeleton to the cytoskeleton. Of particular significance, αSpII plays a critical role in DNA repair where it acts as a scaffold recruiting repair proteins to sites of damage, enabling DNA repair to take place [44, 50].

αSpII’s stability within the cell is critical for all of its functions. It is sensitive to cleavage by the protease, μ-calpain, and excessive activation of μ-calpain activity leads to increased cleavage of αSpII and loss of its ability to function in essential cellular processes, which is a contributing factor in a number of disorders [44, 50]. Maintaining a balance between activation of μ-calpain and regulation of spectrin cleavage is thus extremely important. This is particularly critical during the repair of DNA interstrand crosslinks (ICLs), where αSpII plays a critical role in repair of damaged sites, on both telomeric and non-telomeric DNA [46]. Excessive activation of µ-calpain activity leads to cleavage and breakdown of αSpII and loss of its ability to function in DNA repair as well as in cellular systems critical for both the structural and non-structural components of cell function [69]. This, in turn, leads to development of the pathological changes observed in numerous disorders.

One disorder in which there is a deficiency in αSpII is the bone marrow failure disorder, Fanconi anemia (FA), a distinctive hallmark of which is a defect in ability to repair DNA ICLs. αSpII levels in FA cells are 35%–40% of normal and levels of repair are 35–45% of normal [43, 48, 54]. Of particular significance, loss of αSpII in FA cells is due to increased µ-calpain activity, which is 3-4 fold greater than in normal cells, as observed in FA-A, FA-C, FA-D2, FA-F and FA-G cells [69]. This leads to increased cleavage of αSpII and decreased DNA ICL repair capabilities in both telomeric and non-telomeric DNA (Figure 11A). However, when levels of µ-calpain are knocked down, there is decreased cleavage of αSpII, whose levels return to those found in normal cells, and restoration of DNA ICL repair in these cells (Figure 11B). Another characteristic hallmark of FA is chromosome instability. After DNA ICL damage, a number of chromosomal aberrations occur in FA cells such as sister chromatid end-to-end fusions, chromatin breaks, chromosome exchanges and radials [45]. In FA cells in which µ-calpain has been knocked down, αSpII stability is restored leading to restoration of chromosomal stability and a decrease in chromosome aberrations after DNA ICL damage. This indicates that αSpII plays an important role in both DNA repair and chromosome stability after DNA ICL damage.

Lambert et al. have hypothesized that excessive cleavage of αSpII in FA cells is an important contributing factor in the pathogenesis of this disorder [44, 50]. The strongest evidence supporting this is from the studies noted above on the critical role of αSpII in DNA repair and in chromosome stability, since defects in these two processes are of major importance in FA pathogenesis. αSpII has been shown to play a direct role in DNA ICL repair and in the repair defect in FA. It binds directly to DNA containing ICLS and its involvement in the repair process has been demonstrated both by measurement of unscheduled DNA synthesis (UDS) after damage, which measures uptake of nucleotides into DNA repair patches, and by localization of the repair protein, XPF, to sites of DNA ICLs in normal and FA cells [42, 43, 47]. These studies indicate that breakdown of αSpII in FA cells is directly related to the defect in DNA ICL repair. An important question is what are the factors that are important in maintaining the stability of αSpII in FA cells. Studies have demonstrated that one of these is regulation of cleavage of αSpII by μ-calpain.

Excessive cleavage of αSpII by µ-calpain and its involvement in the DNA repair defect in FA cells has been demonstrated both by the presence of the 150 Kd αSpII breakdown product of µ-calpain in FA cells and by studies which show that knocking down μ-calpain corrects the excessive cleavage of αSpII and the DNA repair defect [69]. This needs to be examined in more FA complementation groups. It will also be important to determine whether there are other proteins involved in αSpII cleavage. Calmodulin, for example, binds to a site on αSpII and can stimulate µ-calpain activity. Studies need to be carried out to determine whether increased binding of calmodulin to αSpII in FA cells plays a role in the increased µ-calpain activity observed and the increased breakdown of αSpII.

FA proteins are known to play a role in the DNA ICL repair pathway. Lambert et al. have hypothesized that they also play an important role, either directly or indirectly, in maintaining αSpII stability. Transfection of FA cells (FA-A, FA-C, and FA-G) with the corresponding FANC-cDNA, which results in expression of either FANCA, FANCC or FANCG, respectively, restores αSpII stability and leads to restoration of chromosomal stability [69]. FANCG has been shown to directly bind to the SH3 domain in αSpII [101]. It has been proposed that FANCG may be involved in aiding in maintenance of αSpII stability by directly preventing binding of low molecular weight phosphotyrosine phosphatase (LMW-PTP) to its SH3 domain, which would lead to dephosphorylation of residue Y1176 at μ-calpain’s cleavage site adjacent to the SH3 domain (Figure 1) and allow µcalpain to cleave αSpII at this site [101]. Binding of FANCG to the SH3 domain would aid in preventing cleavage of αSpII by μ-calpain. This needs to be further investigated. Other FA proteins could also interact with αSpII to protect it from cleavage by µ-calpain or bind to µ-calpain or calmodulin and prevent their activity, either directly or indirectly, via other proteins. FANCA and FANCG, for example, have been shown to bind directly to μcalpain [13, 50]. Whether this binding can decrease the activity of µ-calpain and enhance αSpII stability needs to be examined. Similarly, binding of a FA protein to calmodulin could inhibit its ability to bind to μ-calpain and activate it, which would also enhance αSpII stability. Thus, binding of FA proteins to αSpII or to associated proteins such as μcalpain or calmodulin could have an important effect on αSpII stability, protecting it from cleavage by µ-calpain. Studies need to be carried out on all of these possibilities.

Numerous studies have been done on the direct role FA proteins play in repair of DNA ICLs. In one of these, knockdown of the FANCA and FANCD2 genes in human embryonic stem cells was shown to lead to a marked reduction in localization of FANCA and FANCD2 proteins to ICL damage induced foci, indicating presence of a defect in DNA ICL repair [192]. Chromosomal aberrations such as breaks and radials also formed [192]. It would be interesting to determine, in studies such as these, whether a decrease in FANCA or FANCD2 proteins leads not only to direct effects on DNA ILC repair but also to loss of stability and breakdown of αSpII. If this occurred, it would suggest that loss of the FA protein led to increased breakdown of αSpII and to the increased defect in DNA repair and chromosome stability observed after DNA ICL damage. These studies would also emphasize the importance of FA proteins in aiding in maintenance of the stability of αSpII in cells, as has been suggested.

Another distinctive hallmark of FA is chromosome instability. The importance of αSpII in maintenance of chromosome stability is seen in studies in which knock down of αSpII in normal cells leads to production of chromosomal aberrations [45]. In addition, in FA cells when levels of αSpII are returned to normal by knocking down μ-calpain, chromosome stability is restored indicating that αSpII plays a role in this process [45]. The effect that excessive cleavage of αSpII in FA cells has on chromosome stability could be directly related to the defect in the ICL repair process and the increase in un-repaired DNA which is produced in these cells. It has been shown that un-repaired or misrepaired lesions in DNA can lead to chromosomal aberrations [164]. Cells are particularly sensitive to un-repaired DNA ICLS, which can lead to DNA double-strand break formation in S phase of the cell cycle and to chromosomal aberrations [164]. In FA cells, breakdown of αSpII could thus be important in production of the chromosomal aberrations observed, which particularly occur after DNA ICL damage and which are not effectively repaired in these cells.

Lambert et al. have also hypothesized that excessive cleavage of αSpII in FA cells may be an important contributing factor in some of the other characteristics of FA pathogenesis such as bone marrow failure, development of AML, congenital anomalies and neurological abnormalities [13, 44]. Studies have shown, for example, that there is a loss of αSpII in bone marrow of patients with AML and it has been proposed that this loss may be contributing to AML development and to leukemogenesis [165, 167]. It can be hypothesized that since FA patients can develop AML, a deficiency in αSpII is a contributing factor in its development. Excessive cleavage and loss of αSpII also occurs in a number of neurodegenerative disorders. This is due to excessive activation of calpain and is a common feature of the neuropathology of many of these disorders. Neurological abnormalities are also a characteristic of FA patients [67, 168, 183–186]. It is possible that excessive calpain activity may thus be an important factor contributing to the loss of αSpII and to a number of the neurological anomalies observed in FA. FA could thus represent another disorder in which there is excessive cleavage of αSpII by μ-calpain. Studies needs to be carried out on this in more complementation groups of FA. Since αSpII is a multifunctional protein, it is not surprising that a deficiency in it could lead to deficiencies in multiple cellular pathways and result in a number of the different pathological manifestations observed in FA.

This review has focused on the important role of αSpII in repair of DNA ICLs in telomeric and non-telomeric DNA and on the importance of maintaining the stability of αSpII not only for DNA repair but also for other cellular processes. Two factors which have been shown to be critical for maintaining αSpII stability in the cell are the levels of μ-calpain activity and the presence of FA proteins. Excess μ-calpain activity in FA cells has been demonstrated to lead to breakdown of αSpII. FA proteins are proposed to play a role in regulation of αSpII stability by modulating its cleavage by μ-calpain. Direct binding of FANCG to αSpII has been demonstrated but the effects this binding has on αSpII stability and its cleavage by μ-calpain needs to be further investigated as does the effect of other FA proteins on this process.

Since αSpII has a wide spectrum of functions in a number of different cellular and physiological processes, it is possible that in FA cells excessive cleavage of αSpII by μ-calpain could have an important effect on some of the clinical characteristics of this disorder. Decreasing μ-calpain activity in FA cells could aid in stabilization of αSpII and be of potential therapeutic relevance. Lambert et al. have shown that knocking down μcalpain activity in FA cells to levels found in normal cells does not have any adverse effects on cell survival and it leads to restoration of αSpII to normal levels and enables DNA ICL repair to proceed. Though levels of μ-calpain have been reduced to normal in these cells, the deficiency in the FA protein specific for the FA complementation group being examined has not been corrected. However, DNA repair takes place. FANCD2-Ub is not present but non-Ub FANCD2 is and it localizes in foci at sites of damage. αSpII is needed for this to occur [49]. αSpII localizes in foci at sites of damage along with XPF but not with non-Ub FANCD2. It has been proposed that this could be a backup system in which non-Ub FANCD2 is involved in the repair process [49]. In these FA cells, if a specific FA protein is not available to help stabilize αSpII, restoration of αSpII to normal levels by knocking down μ-calpain could, to some extent, counteract this need for the FA protein in this process. Whether this holds true for all of the FA complementation groups needs to be examined.

It is hypothesized that there is a mechanistic link between excessive μ-calpain activity, cleavage of αSpII, and defective DNA ICL repair, and that these are key factors in the pathogenesis of FA. FA proteins are proposed to play a role in modulating αSpII cleavage by μ-calpain. Correcting the excessive cleavage of αSpII in FA cell is particularly important since αSpII is essential for a number of critical cellular and physiological processes, such as DNA repair. These studies suggest a new direction for correction of a number of the phenotypic deficiencies in FA cells. Thus, stabilization of αSpII in FA by reducing μ-calpain activity, potentially in combination with other modalities, could potentially reduce the DNA repair defect and other systemic deficiencies which lead to the clinical manifestations of this as well as other disorders.