Torsin 1A Interacting Protein 1
Lamina associated polypeptide 1 (LAP1) was identified in 1988 as an integral inner nuclear membrane (INM) protein associated with lamins. LAP1 family members were identified using a monoclonal antibody against rat liver nuclear envelope (NE) extracts (Senior and Gerace 1988). This antibody recognized three polypeptides with apparent molecular weights of 75 KDa, 68 KDa, and 55 KDa in rat liver NEs that were subsequently termed LAP1A, LAP1B, and LAP1C, respectively. The evidence that LAP1 was connected to nuclear lamina was achieved by biochemical extraction experiments, and subsequent in vitro experiments demonstrated that LAP1A and LAP1B bind directly to lamins A, C, and B1 and most likely indirectly to chromosomes (Foisner and Gerace 1993). Further co-immunoprecipitation studies demonstrated that LAP1A/LAP1C co-immunoprecipitated with B-type lamins (Maison et al. 1997).
The first full-sequenced cDNA of LAP1 was determined in 1995 and was referent to rat LAP1C. Partial characterization of the other cDNAs suggested that the three rat LAP1 family members result from alternative splicing, given that LAP1A and LAP1B were identical to the LAP1C sequence, having only two insertions. Subsequently further characterization of LAP1C was achieved. It was shown that LAP1C is a type II transmembrane protein, containing a nucleoplasmic N-terminal domain, a single transmembrane (TM) domain, and a luminal C-terminal domain located in the perinuclear space (Martin et al. 1995).
The first human full-length cDNA of LAP1 was only reported in 2002. LAP1B was isolated from an expression library of HeLa cells and presented around 74% amino acid identity with the predicted rat LAP1B. Further, human LAP1B comprises a single TM domain, and interestingly the lumenal and the nucleoplasmic domain present 89% and 66% amino acid identity with the predicted rat LAP1B, respectively (Kondo et al. 2002). Further, a splice variant of LAP1B was also reported (Tadokoro et al. 2005). Recently, a novel human LAP1 isoform was identified and named LAP1C. This novel human isoform was shown to be present in both human cell lines and tissues (Santos et al. 2014). Both human LAP1B and LAP1C are integral proteins of the inner nuclear membrane (INM).
Human TOR1AIP1 Gene, Transcripts, and Isoforms
The physiological relevance of the LAP1 different isoforms is unknown but some ideas are emerging. LAP1 is ubiquitously expressed in neuronal and nonneuronal tissues (Goodchild and Dauer 2005). Moreover, LAP1A, LAP1B, and LAP1C are detected in liver, spleen, brain, and kidney rat tissues, but LAP1A seems to be the major isoform present in rat liver. LAP1C is the major isoform detected in rat-cultured cells (Senior and Gerace 1988). In human cell lines, this is also true, being that LAP1C is more abundant than LAP1B. However, in human tissues, the expression of LAP1B/LAP1C ratio was found to be tissue specific. Briefly, the expression of both human LAP1B and LAP1C was quite similar in both testis and ovary, and this is in sharp contrast with other tissues like the lung, kidney and spleen, where LAP1C was more abundant than LAP1B. Interestingly, the expression of human LAP1B was higher than LAP1C in the brain and heart (Santos et al. 2014).
A second TOR1AIP1 mutation was reported in a boy, born from consanguineous healthy parents (“Moroccan” mutation), who developed rapidly progressing dystonia, progressive cerebellar atrophy, and dilated cardiomyopathy (Dorboz et al. 2014). The patient’s whole exome sequencing revealed a homozygous missense mutation (c.1448A>C) in the TOR1AIP1 gene that results in the change of a glutamic acid to an alanine at amino acid position 482 (p.E482A) (Dorboz et al. 2014; Rebelo et al. 2015a) (Fig. 2). Of note, this glutamic acid is a highly conserved residue. Using the patient’s fibroblasts, the authors demonstrated that this individual presented reduced LAP1 levels and altered subcellular distribution. A strong reduction of LAP1 at the NE was observed with a concomitant mislocation and aggregation of LAP1 at the endoplasmic reticulum (ER) (Dorboz et al. 2014). Of note, given the position of the mutation, apparently both LAP1 human isoforms (LAP1B and LAP1C) are affected.
Additionally, two novel pathogenic TOR1AIP1 mutations have been recently reported (Ghaoui et al. 2016). In this case, two siblings from an Australian family (“Australian” mutation) revealed severe cardiac failure, musculoskeletal weakness, and muscular dystrophy. The whole genome sequencing of the affected siblings showed a frameshift mutation in exon 1 (c.127delC, p.Pro43fs*15) resulting in a truncated protein of 58 amino acids, lacking the TM domain, and is putatively nonfunctional (Fig. 2). Consequently, no LAP1B is observed in these patients. The second mutation is a missense change in exon 10 (c.1181T>C, p.L394P) that is predicted to be very pathogenic (Fig. 2a) (Ghaoui et al. 2016). Given the position of this mutation, one can speculate that both LAP1 isoforms (LAP1B and LAP1C) are mutated. However, it does not affect LAP1B directly, since it is absent as a consequence of the first mutation. This is not the case for LAP1C which might be expressed and consequently affected by the second mutation. However, these speculative ideas regarding LAP1C expression and mutation are very exciting, but experimental validation is still required.
Functionally Associated LAP1 Interactors
The nuclear lamina is a protein meshwork that lies in the NE and is composed by polymeric lamins that comprises a group of family members of the intermediate filament proteins. It is well accepted that the formation of lamin polymers at the NE is due to the self-association properties of lamins. However, this process may be modulated by lamin-binding proteins of the INM and/or by chromatin (Schirmer and Foisner 2007). LAP1 was one of the first lamina-associated polypeptides described. Moreover, it was shown that LAP1 binds directly to lamin A/C and lamin B (Foisner and Gerace 1993; Maison et al. 1997). In interphase cells LAP1 is located in the INM in association with lamins (directly) and chromosomes (indirectly). The interaction between LAP1 and lamins seems to be important for targeting lamins to the NE and thereby contributing to the maintenance of the NE architecture (Fig. 3b). However, when cells enter mitosis, the NE is disassembled during prometaphase, and LAP1 seems to be redistributed throughout the ER, losing its association with chromosomes and lamins, thereby contributing to the NE breakdown in the early mitotic events and for NE reassembly upon mitosis.
TorsinA, a member of a family of AAA+ proteins (ATPase associated with different cellular activities) localized in the lumen of the ER, interacts with LAP1 (Goodchild and Dauer 2005). A deletion of a single glutamic acid within torsinA is responsible for DYT1 dystonia, a movement disorder characterized by prolonged involuntary twitching movements (Ozelius et al. 1997). Of note, whereas wild-type torsinA is located in ER and perinuclear space, the mutant torsinA is preferentially located in the perinuclear space (Goodchild and Dauer 2004). Further in vivo studies using both torsinA knockout mutant mice and mice with a germline deletion of Tor1aip1 gene revealed neuron-specific nuclear envelope abnormalities suggesting that both proteins are functionally associated in neurons (Goodchild and Dauer 2005; Kim et al. 2010).
Additionally it is known that torsinA is inactive and requires the stimulation of one or two distinctly localizing transmembrane cofactors, LAP1 or LULL1 (luminal domain-like LAP1), that are regulators of torsinA ATPase activity (Zhao et al. 2013). Recently it was shown that LAP1 adopts a partial AAA+ domain fold and co-assembles with torsinA subunits of an active enzyme. These domains comprise a strictly conserved arginine residue that aligns perfectly with a key catalytic arginine found in AAA+ proteins. The latter residue is therefore critical for torsinA ATPase activity (Brown et al. 2014; Sosa et al. 2014). These results confirm both LAP1 and LULL1 as members of the torsin core machinery.
Several lines of evidence suggest that torsinA and LAP1 might have a combined critical role at the NE dynamics (Fig. 3d). Essentially, several hypothetical models of modes of action were pointed out by Laudermilch and Schlieker (2016). Briefly, the recently described nuclear pore-independent vesicle-driven RNA export pathway (budding pathway through the NE) might require the action of both torsin and LAP1. Additionally, torsinA and LAP1 might also mediate the assembly and disassembly of the LINC complex, NE reassembly upon mitosis or upon NE rupture (Laudermilch and Schlieker 2016). These proposed models are extremely interesting and should be further investigated.
Emerin is an INM protein associated with the X-linked form of Emery-Dreifuss muscular dystrophy, which is characterized by progressive skeletal muscle wasting, contractures, and cardiomyopathy (Bione et al. 1994). Several disease-causing mutations in the emerin gene have been identified, and most of them resulted in decreased levels of emerin and its mislocalization in the cytoplasm (Manilal et al. 1998). Emerin was identified as a LAP1 interacting protein upon a screen for identifying novel LAP1-binding proteins (Shin et al. 2013). The interaction of both proteins is mediated by their nucleoplasmic domain, and emerin binds to LAP1 at residues 1–330 (Shin et al. 2013). These authors conditionally deleted LAP1 from mice striated muscle, which lead to the development of profound muscular dystrophy and cardiomyopathy that culminates with premature mice death. Further studies performed by the same authors indicated that LAP1 is essential for normal localization of emerin and A-type lamin complexes in the INM. Taken together these results suggest that LAP1 is a crucial protein for skletal muscle maintenance, probably together with emerin (Fig. 3a). Surprisingly, studies performed with mice lacking emerin presented only few skeletal muscle abnormalities, while emerin loss in humans causes myopathy and cardiomyopathy. Interestingly, the “Turkish” mutation in the gene encoding LAP1 leads to LAP1B absence and consequently to a form of muscular dystrophy. The same seems to occur with the “Australian” mutation. Together, these studies strengthen the hypothesis that the complex LAP1/emerin is functionally associated in skeletal muscle. It would be particularly interesting to determine whether the two human LAP1 isoforms (LAP1B and LAP1C) are differently expressed in specific pathological conditions, since one can deduce that LAP1C also binds to emerin (Rebelo et al. 2015a).
Protein Phosphatase 1
It was recently reported that LAP1B binds to PP1 (Santos et al. 2013). PP1 is the major eukaryotic serine/threonine phosphatase, and it is estimated that it dephosphorylates around one third of all eukaryotic proteins. PP1 is involved in many cellular processes, and these are dictated by the interaction of the PP1 catalytic subunit with different regulatory subunits. More than 200 PP1 interacting/regulatory proteins have been described. The interaction between LAP1 and PP1 was mapped in the nucleoplasmic domain, and it was determined that LAP1 is a PP1 substrate (Fig. 3c) (Rebelo et al. 2015b; Santos et al. 2013). However, the physiological relevance of this has yet to be determined. Additionally, five different LAP1 phosphorylated residues were identified: Ser143, Ser216, Thr221, Ser306, and Ser310. From those, it was possible to determine that PP1 is able to dephosphorylate Ser306 and Ser310 (Santos et al. 2014). Recent studies also indicate that LAP1 is highly phosphorylated during mitosis (Fig. 3c) (Santos et al. 2015), and given the importance of PP1 during the cell cycle, the physiological relevance and association of these two proteins deserve further elucidation. In particular, the hypothesis that LAP1 phosphorylation might represent a key regulatory mechanism across the nuclear membrane is extremely attractive from a functional perspective at a molecular level.
Novel Functionally Relevant Putative LAP1 Interactors
Functionally relevant putative LAP1 interactors are emerging and will be discussed in this section. The identification of novel LAP1 interacting proteins and their associated cellular functions seems a very important strategy to understand the precise physiological roles of LAP1. Recently, the LAP1 interactome was explored and 41 LAP1 interactors were identified. Of note, this bioinformatic study not only reinforces the putative functions already ascribed to LAP1 but importantly novel putative functions that are associated to the novel putative interactors were reported (Serrano et al. 2016a). Briefly, from the analysis of the biological processes associated to the LAP1 interactome, a significant enrichment in the following was observed: regulation of response to DNA damage, nuclear membrane organization, nuclear envelope organization, cell cycle, chaperone-mediated protein folding, nucleus organization, telomere maintenance, cellular component organization, telomere organization, and cellular component disassembly involved in execution phase of apoptosis (Serrano et al. 2016a). From these biological processes, the regulation of response to DNA damage, telomere maintenance, and telomere organization had never before functionally associated to LAP1. LAP1 seems to be associated with the shelterin complex, which is a multiprotein complex with DNA remodeling activity that promotes structural changes of the telomeric DNA protecting chromosomes ends. The shelterin complex is therefore a protective complex which prevents the recognition of telomeres as sites of damage and is composed of six telomere-specific proteins (TRF1, TRF2, POT1, TIN2, TPP1, and TRF2IP1). Of note, disruption of the shelterin complex leads to activation of DNA damage, a response that includes H2A.X (histone 2A family member) and ATM (ataxia telangiectasia mutated) phosphorylation (Arnoult and Karlseder 2015; de Lange 2005). Given the emerging functions of LAP1 and the identification of novel putative binding partners, TRF2, TRF2IP1, RIF1, and ATM, the hypothesis that LAP1 might have a role in assembly or stabilizing some of the proteins of the shelterin complex is very attractive. Further, bioinformatic studies using the Ingenuity Pathways Analysis tool also strengthened this hypothesis. Interestingly, two canonical pathways associated with the LAP1 interactome were identified: telomerase signaling and telomere extension by telomerase (Serrano et al. 2016a).
Previous studies suggested a putative function for LAP1 related with the cell cycle. Remarkably, in the bioinformatic study of the LAP1 interactome, the cell cycle was in the top three biological processes identified. Neumann et al. reported that LAP1 might have a role in the assembly of mitotic microtubule spindle and consequently in mitosis progression, since the knockdown of LAP1 caused impaired microtubule spindle formation during prometaphase that culminates with aberrant mitotic exit (Neumann et al. 2010). Additionally, LAP1 co-localizes with acetylated α-tubulin in the mitotic spindle and with γ-tubulin in centrosomes (main microtubules organizing center) in mitotic cells. The knockdown of LAP1 results in a decreasing number of mitotic cells, and the positioning of the centrosome near the NE was also altered (Santos et al. 2015). Moreover, the association between MAD2L1 and MAD2L1BP occurs in the mitotic checkpoint complex (MCC). Both proteins also bind to LAP1 and MAD1L1 (Hutchins et al. 2010). Interestingly, the MCC inhibits the anaphase-promoting complex/cyclosome (APC/C), an ubiquitin E3 ligase that promotes chromosome segregation. This inhibition regulates the spindle assembly checkpoint (SAC) that is responsible for the inhibition of chromosome segregation until all sister chromatids achieve bipolar attachment to the mitotic spindle. All together these studies place LAP1 as a key protein in the regulation of cell cycle progression and functionally associated to the MCC.
Another recently described LAP1 function is related with spermatogenesis. We recently determined that LAP1 is expressed during nuclear elongation in spermiogenesis, and it is located at the centriolar pole of spermatids (Fig. 3e). LAP1 is also identified as a novel crucial element for manchette development and a new chromatin regulator via lamin interaction. Overall, by moving to the posterior pole, LAP1 can contribute to the achievement of nonrandom, sperm-specific chromatin distribution and can modulate cellular remodeling during spermiogenesis (Serrano et al. 2016b).
Lamina-associated polypeptide 1 (LAP1) is a type II transmembrane protein of the inner nuclear membrane encoded by the TOR1AIP1 gene. Three LAP1 isoforms have been reported in rat, as a result of alternative splicing. However, in humans only two isoforms were reported, namely, LAP1B and LAP1C, both being abundant in human cells and tissues. Their specific function and mode of action are unknown and deserve further investigation. Several LAP1 binding partners have been asserted; hence some functions have been ascribed to LAP1. These binding partners include lamins, torsinA, emerin, and protein phosphatase 1; and together they accomplish crucial cellular functions, namely, nuclear architecture maintenance and chromatin regulation (LAP1-lamins), regulation of torsinA ATPase activity, nuclear envelope localization of torsinA and NE dynamics (LAP1-torsinA), nuclear envelope integrity maintenance and skeletal muscle maintenance (LAP1-emerin), and regulation of cell cycle progression (LAP1-PP1). Recently four mutations in TOR1AIP1 gene were identified and correspond to the “Turkish” mutation, “Moroccan” mutation, and “Australian” mutation, the latter consists of two different mutations. This new data increases the number of genes associated with nuclear envelopathies and strengthens the hypothesis of LAP1 as a key protein for skeletal and cardiac muscle function and also brain function.
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