Clinic Rev Allerg Immunol (2012) 42:58–70 DOI 10.1007/s12016-011-8290-y

Autoimmune Diseases and Polyamines Wesley H. Brooks

Published online: 26 November 2011 # Springer Science+Business Media, LLC 2011

Abstract Genetics and environmental factors have important roles in autoimmune diseases but neither has given us sufficient understanding of these mysterious diseases. Therefore, we are now looking closer at epigenetics, an interface between genetics and environmental factors. Epigenetics can be defined as reversible heritable changes to chromatin that can alter gene expression without altering the gene’s DNA sequence. Methylation of DNA and histones are primary means of epigenetic control. By adding methyl groups to DNA and histones, it can limit accessibility of the underlying gene thereby altering the amount of gene expression. The methyl group is derived from an essential molecule in the cell, S-adenosylmethionine (SAM). However, a group of small molecules called polyamines also require SAM for their synthesis. Polyamines are essential for many cellular functions and polyamine activity is increased in many autoimmune diseases. Presented here is the “polyamine hypothesis” in which increased polyamine synthesis competes with cellular methylation (epigenetic control) for SAM. It is proposed that increased polyamine activity can cause disruption of cellular methylation, which can lead to abnormal expression of previously sequestered genes and disruption of other methylationdependent cellular processes. Keywords Autoimmune diseases . Polyamines . LINE1 . Alu . X chromosome . Post-translational modifications

W. H. Brooks (*) Experimental HTS, SRB-3, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Dr., Tampa, FL 33612-9416, USA e-mail: [email protected]

Abbreviations AID Autoimmune disease(s) AMD SAM decarboxylase, a key enzyme in polyamine synthesis (a.k.a. SAMDC) dcSAM Decarboxylated S-adenosylmethionine ODC Ornithine decarboxylase PAD Peptidylarginine deiminase PAO Polyamine oxidase PAR1 Pseudo-autosomal region 1 of the X chromosome RA Rheumatoid arthritis SAM S-Adenosylmethionine, the cellular methyl group donor SjS Sjögren’s syndrome SLE Systemic lupus erythematosus SMS Spermine synthase SSAT Spermidine/spermine-N1-acetyltransferase (a.k.a. SAT1) XCI X chromosome inactivation XIC X inactivation center Xi Inactive X chromosome

Introduction Autoimmune diseases (AID) present a very complex picture influenced by genetics, epigenetics, sex, age, and environmental factors. Since specific disease-related genetic abnormalities with validated roles in AID have been elusive, interest is growing in epigenetics in AID. One key concept in epigenetics is that methylation of chromatin is a means of reversibly controlling gene expression without altering the underlying gene sequence. Abnormalities in methylation are seen frequently in AID and changes in methylation patterns appear to be critical in disease development [1].

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how autoantigens could arise based on the proposed increased synthesis, recycling and activity of polyamines that adversely impacts methylation.

For example, hydralazine and procainamide, known to cause drug-induced lupus, cause DNA hypomethylation in T cells and autoreactivity [2]. In addition, monozygotic twins discordant for systemic lupus erythematosus (SLE) show notable differences in their DNA methylation patterns [3]. Therefore, we should consider how abnormal methylation changes could occur in AID and what consequences could develop. S-Adenosylmethionine (SAM) is essential in the cell since it is the methyl donor for cellular methylation. However, SAM can be decarboxylated by SAM decarboxylase (AMD) and used in polyamine synthesis (Fig. 1a). Polyamines, primarily spermidine and spermine, and their precursor putrescine are essential in cells for translation, transcription, replication and other functions. Normally, SAM is abundant in cells to support methylation. However, polyamines are elevated in AID [4]. An important role for polyamines in AID has been proposed previously, based on competition between methylation and polyamines for SAM [5, 6]. A particularly intriguing aspect of the hypothesis relates to potential loss of epigenetic control of polyamine genes on the X chromosome that leads to an abnormal increase in polyamine synthesis and recycling, taxing SAM levels and creating higher levels of polyamines and reactive moieties from polyamine oxidation. This X-linked scenario could also help explain the female bias seen in many AID. Here, we review this hypothesis, and present additional details on

Polyamines Polyamine Synthesis and Recycling Polyamine synthesis (Fig. 1b) occurs primarily in S phase and as part of a stress response [7]. Otherwise, polyamine synthesis is tightly controlled by: rapid turnover of RNA and proteins of polyamine enzymes; feedback inhibition; and by an antizyme to ornithine decarboxylase (ODC) [8]. Polyamine synthesis begins with decarboxylation of ornithine by ODC producing putrescine. Putrescine is the precursor for higher polyamines but also, an important point, putrescine binds an allosteric site in AMD, increasing AMD activity eightfold (Fig. 2a, b) [9]. The resulting decarboxylated SAM (dcSAM) and putrescine are then used in synthesis of spermidine by spermidine synthase (SDS) and spermidine and dcSAM are used in synthesis of spermine by spermine synthase (SMS). Due to putrescine’s key role in polyamine synthesis, putrescine is present in eukaryotes only in trace amounts normally to prevent any undesirable drop in SAM levels that could affect methylation. We should note that dcSAM cannot serve as a methyl donor in place of SAM. NH3+ +H N 3

Putrescine

Ornithine SAM

AMD dcSAM

ODC

NH3+ N+ H2

Putrescine +H N 3

SDS Spermidine

SMS B.

+H N 3

Spermine

PAO SSAT

A.

N+ H2

Spermidine N+ H2

NH 3+

Spermine

Acetylspermidine

PAO SSAT

Fig. 1 The polyamine pathway. a The diamine precursor, putrescine, and the main polyamines in eukaryotes: spermidine and spermine. b Polyamine synthesis begins with conversion of ornithine from the urea cycle to putrescine by ornithine decarboxylase (ODC). Putrescine binds an allosteric site in S-adenosylmethionine decarboxylase (AMD), which converts S-adenosylmethionine (SAM) to decarboxylated SAM (dcSAM). Putrescine is converted to spermidine by spermidine

Acetylspermine

synthase (SDS) using dcSAM. Spermidine is converted to spermine by spermine synthase (SMS), also using dcSAM. Recycling/salvaging of polyamines involves acetylation of spermine and spermidine by spermidine/spermine-N1-acetyltransferase (SSAT) to create acetylspermine and acetylspermidine, respectively. These are then oxidized by polyamine oxidase (PAO) to create spermidine and putrescine, respectively

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B. A. Active Site

Allosteric Site

Fig. 2 S-Adenosylmethionine decarboxylase. a S-Adenosylmethionine decarboxylase (AMD, a.k.a. SAMDC) showing the active site with inhibitor MeAdoMet and the allosteric site with putrescine (file: 1I7B.pdb in Protein DataBank, www.rscb.org) [113]. Putrescine binding reorients hydrophilic residues through the center of the

enzyme, opening the active site. Putrescine also stabilizes the two units of the self-cleaving/activating enzyme. b AMD’s substrate, SAM, and product, dcSAM. Note that the aminopropyl group, leftmost +H3N(CH2)3 of dcSAM, is used in making spermidine and spermine, leaving methylthioadenosine (MTA)

Polyamines can be recycled (spermine to spermidine, spermidine to putrescine) by acetylation, carried out by spermidine/spermine-N1-acetyltransferase (SSAT), and followed by oxidation by polyamine oxidase (PAO) [10]. This creates the interesting possibility that SSAT/PAO is an alternate route for producing putrescine, bypassing the tightly controlled ODC and keeping AMD and the polyamine pathway active, even wastefully using SAM by looping through polyamine synthesis and recycling [6]. In addition, reactive products (e.g., hydrogen peroxide and acrolein) can be generated from polyamine oxidation and can damage proteins [50]. Acrolein-conjugated proteins are significantly increased in primary Sjögren’s syndrome (SjS), suggesting possible involvement of polyamine recycling enzymes [58]. Excess putrescine and PAO activity are seen in rheumatoid arthritis (RA) [11–13]. On the other hand, increased synthesis of polyamines could increase many of the polyamine interactions with cellular components (described below), and it could shift the ratio of spermidine (+3) and spermine (+4) affecting those interactions in which they may compete. Thus, there is a need to keep polyamine synthesis and recycling under control.

be generated from synthesis or recycling when there is an event that requires free polyamines, as in S phase or cellular stress, where polyamines can help stabilize new or disrupted chromatin and other macromolecular complexes. Typically, spermidine is present at 1.0 to 1.3 mM and spermine at 0.9 to 1.6 mM [14]. In eukaryotes, approximately 60–85% of polyamines are bound to RNA, 4–18% are bound to DNA, 2–6% are bound to phospholipids, 2–12% are bound to ATP, and 2– 15% are free [14]. Note the predominance of polyamine associations with RNA, which includes ribonucleoprotein complexes. Much of RNA synthesis and maturation occurs in nucleoli and so we should expect to find significant amounts of polyamines in nucleoli. Indeed, this has been observed in isolated nuclei [15] and as increased polyamine-conjugated proteins in nuclei and nucleoli, with most putrescine- and spermidine-conjugated proteins found in nucleoli [16]. Also, increased RNA synthesis in nucleoli induced by estradiol (E2) involves increased polyamine synthesis [17]. And so polyamine involvement in nucleoli is another important point to keep in mind.

Cellular Content and Localization of Polyamines

Polyamine Interactions

Most cellular polyamines are bound to macromolecular structures, such as nucleic acids. Free polyamines can

Polyamines have many important roles in cells (Table 1) [14, 18–21]. The combination of length and charge

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Table 1 Polyamine interactions Polyamines and chromatin Spermine aids in chromatin condensation and radioprotection of DNA [22] Polyamines stabilize alternate DNA forms (e.g., Z-DNA), modulating supercoils and loops [23] Polyamines stimulate histone acetylation [24] Polyamines alter conformation of nucleosome core particles [21, 25] Polyamines aid DNA repair [26] Polyamines are involved in DNA replication [27] Polyamines are essential in translation Spermidine-lysine conjugate oxidized to hypusine in translational initiation factor eIF5A [28] Spermidine required for aminoacyl-tRNA-synthetase to charge tRNA [29] Translation efficiency improved with polyamines stabilizing tRNA conformation [30] Polyamines help ribosome assembly and stabilization in nucleolus [29] Polyamines modulate cellular enzymatic events Spermine interacts with ATP-Mg+2 to modulate energy dependent reactions [31] Polyamines can serve as substrate for acetylases and transglutaminases [32, 33] Polyamines provide feedback control on their own synthesis [34] Polyamines and receptor binding Polyamines modulate binding of estrogen receptor to Z-DNA forming sequences [35] Polyamines influence binding of vitamin D receptor and other nuclear receptors [36] Polyamines influence binding of NMDA receptors [37] Polyamines and membranes Polyamines protect membrane phospholipids from peroxidation and affect membrane microviscosity [38] Polyamines influence local ionic milieu by acting on ion channels [39–41] Polyamines aggregate transmembrane proteins, aiding adhesion and complex formation [42] Other interactions of polyamines Polyamines can modulate blood–brain and blood–nerve barriers [43] Polyamine effect on neurotransmitter uptake in synapses [44] Spermidine purported to be a component of myelin sheath [45] Spermine acts as a free radical scavenger [46] Polyamines are involved in apoptosis [47] Spermine and spermidine serve as negative regulators of macrophages in inflammation [48] Spermine stabilizes insulin mRNA [49]

(Fig. 1a) gives them unique possibilities to interact with multiple anionic sites, such as RNA, DNA and phospholipids. Spermine, for example, has a +4 charge spread over ∼16 Å. This allows spermine to help stabilize chromatin structure and condense DNA in the nucleus [22]. In addition, spermine can stabilize DNA in alternate conformations, such as Z-DNA [23], and polyamines stimulate histone acetylation, which can alter epigenetic control of genes [24]. So, the importance of polyamine interactions with chromatin is evidenced by the requirement for polyamines in cell proliferation, DNA replication and repair, and transcription. Polyamines are also essential in translation. For example, polyamines help in assembly and stabilization of ribosomes in nucleoli [29], and polyamines have interactions at the cell membrane since they can bind phospholipids altering membrane viscosity while protecting phospholipids from peroxidation damage [38]. Poly-

amines can also bind cytoplasmic portions of multiple transmembrane proteins, aggregating proteins, as in cell anchorage [42]. In addition, polyamines compete for important cellular components, such as SAM, ATP and acetyl CoA. Oxidation products of polyamines (e.g., hydrogen peroxide and acrolein) can also affect cells [50], and polyamines can be conjugated to proteins by transglutaminases [32, 33]. These interactions of polyamines underline their importance and the need to control their levels. Polyamines in Autoimmune Diseases Table 2 presents some links between polyamines and AID. These reports show polyamine levels are frequently altered in AID. For example, elevated polyamine levels are seen in urine of patients with RA, SLE, and kidney dysfunction in diabetes [12, 55, 66]. Polyamines

62 Table 2 Possible connections between polyamines and autoimmune diseases

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Rheumatoid arthritis Polyamine oxidase activity increased in RA synovial fluid [13] Increased polyamines may downregulate IL-2 production in RA [51] Polyamine levels in synovial tissues and synovial fluids in RA patients [52] IL-1β induces elevated SSAT1 activity and putrescine in synovial adherent cells [11] Elevated levels of urinary polyamines in patients with RA [12] Systemic lupus erythematosus Enhanced binding of lupus sera to polyamine-induced left-handed Z-DNA [53, 54] Elevated polyamines in urine of children with SLE [55] Polyamine inhibitor reverses abnormal thymic T cell development in MRL mice [56] Ornithine decarboxylase suggested as target in autoimmune diseases [57] Sjögren’s syndrome Acrolein, a toxin produced from polyamines, has intense correlation with primary SS [50, 58] Multiple sclerosis Neurodegeneration is related to aldehyde load [59] Polyamines have a large impact during stress on neural homeostasis and integrity [60] Methylthioadenosine, a spermine synthase inhibitor, is in preclinical studies for MS [61, 62] Spermine, a nitric oxide source, can lead to glutamate release and neural death in neuro-diseases [63] Alterations in polyamine levels and ratios in brains of mice with myelin-deficient neurodegeneration [64] Blood–brain barrier breakdown linked to stimulation of ODC activity and polyamine synthesis [65] Diabetes Polyamines provide a means of early detection of diabetic kidney dysfunction [66] Increased polyamine catabolism alters control of glucose metabolism [67, 68] Increased polyamine levels in erythrocytes in diabetes [69] Spermine may contribute to dysfunction in diabetic retina [70] Myasthenia gravis Altered polyamine levels in skeletal muscle of patients with myasthenia gravis [71]

are elevated in skeletal muscles in myasthenia gravis [71], erythrocytes in diabetes [69], and in synovial tissue and fluid in RA [52]. With regards to RA, besides elevated polyamine levels, IL-1β induces elevated SSAT activity and increased putrescine in synovial cells in RA [11], which fits with the increased PAO activity seen in synovial fluid of RA patients [13]. In diabetes, control of glucose metabolism is altered when polyamine catabolism increases [67, 68]. This loss of control of glucose metabolism in white adipose tissue is attributed to continual cycling through the polyamine pathway, wastefully using ATP and acetyl CoA. Also in diabetes, spermine is believed to control calcium channels in microvasculature of the retina and so changes in spermine levels may have a causative role in retinal damage [70]. Strong support for a role of polyamines in AID comes from research on SLE and SLE models. Anti-DNA autoantibodies from SLE patients show significantly increased binding to DNA stabilized by spermine in Z-DNA conformation, suggesting spermine was involved in the original provocation that led to B cell activation [53, 54]. In addition, targeting polyamine synthesis with difluorome-

thylornithine, a known inhibitor of ODC, shows therapeutic potential, at least in mouse models of lupus [56]. In order to provide further support for polyamine involvement in AID, we must first discuss the possible involvement of the X chromosome in AID and the role that may be played in AID by X-linked polyamine genes.

Female Bias of Autoimmune Diseases Among the approximately 80+ AID, most show a predominance of females among sufferers. For example, the female/male ratio for SLE is 9:1, for myasthenia gravis it is 3:1, for RA it is 2:1, and for MS it is 3:2 [72]. This female bias suggests involvement of the X chromosome and possibly the X inactivation process, which is a major epigenetic event. Some reports have pointed to X chromosome involvement in AID. For example, demethylation of a B cell costimulatory gene, CD40LG, has been observed on the inactive X chromosome of female lupus patients, suggesting it may have a role in the female bias of SLE, possibly due to loss of dosage compensation for the gene, resulting in over-expression [73]. Also, lupus symptoms are

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seen on rare occasions in suffers (males) and carriers (females) of X-linked chronic granulomatous disease (XCGD), a disease not normally considered as an AID [74, 75]. X-CGD is attributed to abnormalities in the cytochrome b-245 beta chain gene located at Xp21.2 in the X chromosome short arm (Xp). In this recessive disease, males who inherit the abnormal gene on their single X chromosome cannot generate a burst of oxidase activity in their macrophages to clear bacterial infections. Females are carriers and have a normal copy of the gene on one of their X chromosomes so they can clear infections. In another report, an XX male, with triplication of some genes on the distal end of the Xp due to an Xp22.33;Yp11.2 translocation, had severe SLE [76]. These reports hint at involvement of the X chromosome, and possibly disruption of the epigenetic control normally established through X chromosome inactivation.

X Chromosome Inactivation The Inactive X Chromosome Human female cells normally have two X chromosomes whereas human male cells have only one X chromosome. Most genes on the X are not sex-related and, therefore, should be expressed at similar levels overall in both female and male cells. In order to achieve this equivalency (a.k.a. dosage compensation), one X chromosome in the female cell is inactivated, resulting in an inactive X (Xi, a.k.a. the Barr body) and an active X (Xa) [5]. This process, X chromosome inactivation (XCI), is a major epigenetic event and there are multiple components used for maintaining XCI, including methylation of DNA in gene promoters and methylation of lysine residues in histone H3 [5, 77]. The non-coding X Inactivation Specific Transcript (XIST) RNA is the primary factor involved in initiating and spreading the X inactivation state, starting from the X inactivation center (XIC) at Xq13 on the long arm of the X and spreading along contiguous chromatin until most of the Xi is coated with XIST RNA. The XIST RNA associates with the Xi and recruits methyltransferases that instill the epigenetic silencing. The resulting Xi takes on a dense heterochromatic state. The Xi locates to the nuclear periphery and, as a result, the Xi replicates later in S phase than the other chromosomes. The Xi is often found in association with nucleoli, which are believed to have a role in DNA repair and packaging of the Xi [78]. Due to the Xi’s different temporal and spatial treatment relative to other chromosomes, and its need for extensive methylation and packaging, maintaining the Xi can be problematic when a cell is stressed. Most of the Xi genes are inactivated but a variable amount, primarily in the short arm (Xp), distant from the

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XIC, escape inactivation, so that we can think of typically 75–85% of Xi genes being inactive [79]. The extent of XCI varies from cell to cell, from individual to individual, and can decrease with age after trying to maintain the extensive heavy Xi methylation through multiple cell cycles and stresses when cells attempt to pass on their epigenetic patterns to daughter cells [80]. This implies that formerly suppressed X-linked alleles could become active and lose their dosage compensation, as exemplified by the X-linked CD40LG gene mentioned above [73]. The Inactive X Chromosome and X-linked Polyamine Genes What is very intriguing about possible involvement of polyamine synthesis and recycling in AID is that the SSAT and SMS genes are located at Xp22.1 on the short arm of the X chromosome. This could provide an explanation for female predominance in AID. SSAT and SMS are normally expressed from the Xa and they are inactive on Xi in females, but they are surrounded by other genes that are active on the Xi [79, 81]. If epigenetic control of SSAT and/ or SMS on the Xi were lost (i.e., loss of dosage compensation), then over-expression could result in increased polyamine synthesis by SMS and recycling by SSAT, wastefully producing polyamines only to then recycle them, in the process needlessly using SAM, thereby hampering methylation [6, 82]. The late replication of the Xi relative to other chromosomes, its peripheral location in the nucleus, and its heavy requirement for methylation to maintain the inactive state means that, relative to other chromosomes, the Xi is potentially more vulnerable to decreases in SAM levels that could result in loss of methylation patterns due to cellular stress or aging [5]. Recurrent stresses could lead to accumulation of hypomethylation sites, particularly in vulnerable chromatin (e.g., Xi) with loss of epigenetic control, including loss of dosage compensation of SSAT and/or SMS. Then the additional impact on SAM levels from increased polyamine synthesis and recycling could reach a threshold that leads to rapid global hypomethylation with over-expression of other genes. In the case of the Xi, it is not just methylation of DNA and histones that is problematic, but also the scaffold attachment factor, SAF-A, and other proteins that must be methylated in a timely manner to translocate into the nucleus where they help condense the Xi. We should note that this proposed loss of suppression of SSAT and/or SMS on the Xi by repeated insults to the methylation status of the Xi is only one way in which polyamine synthesis and recycling could become over active. The enzymes, at least from the Xa, do have some basal expression and activity and they can be induced in a

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controlled manner. Alternatively, some pathogens induce increased polyamine synthesis and that could be significantly stressful for the cell. For example, Epstein-Barr virus, which has been associated with many diseases including some AID [83], stabilizes the host cell’s c-MYC [84]. Approximately 15% of genes in the human genome are influenced by c-MYC, among which is ODC [85]. This induction of ODC could lead to elevated levels of putrescine, which keeps the allosteric site of AMD occupied, leading to decreased levels of SAM, increased hypomethylation, and excessive polyamine activity. This could in itself lead to the excess polyamine activity that is damaging, particularly with heavy viral activity in the cell, or it could be one of many cellular insults that eventually reach a point of no return for the cell to recoup its epigenetic control.

Generation of Autoantigens Polyamines and Nucleoli At this point, we have described the basic concepts of the polyamine hypothesis. It is a scenario in which there are increased polyamines and increased polyamine oxidation products, such as acrolein, that result from over active polyamine synthesis and recycling. This can explain, for example, the acrolein-conjugated proteins seen in SjS [58] and the polyamine-conjugated proteins seen in nucleoli could be increased [16]. We can extend the hypothesis further to explain many of the autoantigens in AID. We have mentioned the importance of polyamines in the nucleoli, such as in assembly of ribosomes [29]. It is in the nucleoli that much of the maturation of RNA transcripts and ribonucleoprotein assembly occurs and some of the autoantigens seen in AID are involved in RNA transcript maturation, such as the Ro and La proteins, which are found in the nucleoli [86, 87]. The ability of polyamines to interact with nucleic acids and stabilize alternate conformations suggests that increased polyamines could be interfering with nucleoprotein assembly, such as in the nucleoli. It is during cellular stress that the nucleoli become particularly active [100]. In addition, polyamine enzymes are induced during cellular stress and polyamines increase activity in the nucleoli [17]. In this scenario of increased polyamine activity, there is the potential for lower SAM levels that impact cellular methylation. This loss of integrity of chromatin could lead to expression of previously sequestered genes. And lower SAM levels could also affect methylation involved in cellular signaling and intracellular localization of RNA and proteins, such as the scaffold attachment factors that help to condense the Xi.

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Autoantigenic Proteins and Ribonucleoprotein Complexes Approximately 13% of the human genome consists of Alu elements, which are believed to have spread via retrotransposition events. Alu elements range from 80 to 410 bases long and are remnants of the Alu domain of the signal recognition particle (SRP) [88, 89]. Alu elements are very CpG rich and are mostly methylated. Alu elements account for only 8% of the X chromosome, however, there is a very large concentration of them (28.9%) in the pseudoautosomal region 1 (PAR1) of the short arm of the X (Xp) (Fig. 3a, c) [90]. Alu elements can be transcribed by RNA polymerase III (RNA pol III) and many of the RNA transcripts that are assembled into ribonucleoprotein complexes in the nucleoli are also transcribed by RNA pol III, such as U6 RNA, tRNAs and 7SL RNA [91]. Some RNA pol III activity occurs in perinucleolar compartments where transcripts can accumulate [92]. As was mentioned before, the Xi is usually found in close association with nucleoli [78]. This places the dense cluster of Alu elements in PAR1 near the RNA pol III activity of the nucleolus. Consider the possible consequences if many Alu elements were suddenly expressed due to hypomethylation. These Alu transcripts could compete with the SRP, which is assembled in the nucleolus, for SRP9 and SRP14 proteins that normally bind exclusively to the Alu domain of the 7SL RNA in the SRP. Figures 4 and 5 discuss this potential problem. In effect, interference of SRP assembly/export and interference with the translation pause of the ribosome by the SRP Alu domain due to missing SRP9/14 proteins means extracellular proteins could be expressed in the cytoplasm and exposed to intracellular enzymes, such as peptidylarginine deiminase (PAD), which converts arginine residues to citrulline. This could be the source of citrullinated proteins seen in AID, such as citrullinated myelin basic protein in multiple sclerosis. Other abnormal modifications could also occur (ex. acrolein conjugation) and these could interfere with subsequent glycosylation and other normal posttranslation modifications that should occur on the extracellular proteins in the endoplasmic reticulum. Also note that autoantibodies targeting Alu RNA/protein complexes have been found in SLE [93]. Autoantigenic DNA Approximately one third (34%) of the X chromosome consists of LINE1 elements [90]. This is twice the amount found in other chromosomes (17%). The content of LINE1 is greater in the long arm, Xq, and drops in the short arm, Xp, towards the terminus (Fig. 3b). This is consistent with the spread of XCI and the XIST RNA originating from Xq13, suggesting a role for LINE1 in the XCI process [94]. Recently, it has been found that “young” LINE1 elements

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PAR1

<7% LINE1 SMS SSAT

XAR

Xp22.1

Xp

28.9% Alu

‘young’ LINE1 elements Average 8% Alu in X

Centromere XIC XCR

Average 34% LINE1 in XCR

Xq

PAR2 X structure

LINE1 content

A.

Alu content

B.

C.

Fig. 3 X chromosome characteristics. a Xp short arm, Xq long arm, XAR X-added region (chromatin added since divergence of X and Y chromosomes from common ancestral autosome), XCR X-conserved region (chromatin kept in X from ancestral autosome but lost from the Y), PAR1, PAR2 pseudo-autosomal regions (contain some commonality of genes between X and Y), Xp22.1 location of SMS and SSAT genes, XIC X inactivation center (from which silencing of the inactive X initiates and spreads through contiguous chromatin). b LINE1

elements constitute 17% of the genome but make up 34% of the X. The distribution is in a gradient, however, that decreases moving away from the XIC, suggesting a role in X inactivation for LINE1. “Young” LINE1 elements are believed to aid some genes to escape inactivation and some of these LINE1s may still code for functional reverse transcriptases. c Alu elements constitute ∼10% of the genome, but make up only 8% of the X. However, a dense cluster of Alu elements constitute 28.8% of PAR1 (based on Ref. [90])

in the Xp may be actively transcribed in order to keep some regions on the Xi active [95]. Most LINE1 elements, which originated from reverse transcriptases, have lost their

functionality as reverse transcriptases due to mutations and deletions. However, some LINE1 elements still have reverse transcriptase activity [96]. Therefore, these “young”

SRP68

SRP19

‘Alu Domain’

‘Signal Domain’

Signal Recognition Particle

SRP14/SRP9

7SL RNA SRP72 SRP54

A. B.

Signal Recognition

Elongation Pause

Nucleolus mRNA

SRP Signal peptide

Ribosome

CYTOPLASM

Extracellular targeted protein

Nascent polypeptide

Fig. 4 SRP assembly and participation in translation. a The signal recognition particle (SRP) consists of the 7SL RNA and SRP proteins [107, 108]. Assembly occurs in the nucleolus [109]. b The SRP has a signal recognition domain, which binds a signal peptide sequence emerging from a ribosome during translation of extracellular targeted proteins that must, therefore, be translated into the endoplasmic reticulum (ER). The Alu domain of the SRP then binds to halt

Translocon SRP Receptor

ENDOPLASMIC RETICULUM

translation until the ribosome/mRNA/SRP can translocate to the surface of the ER. At the ER, a SRP receptor joins the ribosome to a translocon. The SRP releases to recycle and the ribosome continues translating. Note that the extracellular targeted protein is not exposed to cytoplasmic enzymes or reactive agents since it is expressed into the ER

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SRP68

SRP72

SRP54

SRP19

Incomplete Signal Recognition Particle

7SL RNA

Alu + SRP9/14

A.

SRP14/SRP9

B.

Alu RNA

Nucleolus

mRNA No pause Ribosome Incomplete SRP

Peptide exposed to abnormal modifications

CYTOPLASM

Nascent polypeptide

Translocon SRP Receptor

ENDOPLASMIC RETICULUM

Abnormally modified/folded extracellular protein

Fig. 5 Abnormal SRP Assembly and Translation. SRP9/14 proteins, which are 20× more abundant than the 7SL RNA, dimerize in the cytoplasm and then translocate to the nucleolus [110]. SRP9/14 binds exclusively to the Alu domain [111]. a Excessive expression of Alu elements, such as from the PAR1 Alu cluster on the inactive X chromosome near a nucleolus, could interfere with SRP assembly by competing for SRP9/14. Also, the SRP would have to compete with the Alu elements for export from the nucleolus. b In the cytoplasm, incomplete SRP or a shortage of SRP could allow extracellular targeted proteins to be expressed into the cytoplasm where they could be abnormally modified. Even if an incomplete SRP binds the signal

peptide, it lacks the SRP9/14 proteins to halt elongation. However, it still is capable of translocating proteins to the ER, even without the Alu domain and SRP9/14 [112]. The exposed nascent polypeptide in the cytoplasm could be modified by acrolein-conjugation, polyaminetransglutamination or other enzymes or reactive molecules. In addition, peptidylarginine deiminases (PAD) could convert arginine residues to citrulline (e.g., citrullinated MBP). These abnormally modified proteins could accumulate in the cytoplasm, be degraded or eventually be inserted into the ER. They may suffer misfolding and may receive improper glycosylation due to their abnormalities. Outside of the cell, they could be autoantigenic

LINE1 elements in Xp are suspect for potential reverse transcriptase activity if they have not been extensively mutated yet and are somehow translated into active protein. However, the conditions under which such activity would occur are unknown but appear to involve ribonucleoprotein complexes. LINE-1 reverse transcriptases are particularly adept in handling Alu and U6 RNAs [97, 98]. We should also note that human endogenous retroviruses (HERVs) are found throughout the genome, including the X chromosome, and they have potential reverse transcriptase activity [82, 99]. Alu DNA is over-represented in SLE patients’ sera, constituting 55% of free DNA in sera of SLE patients versus 13% in controls [101]. One may argue this is due to selective protection of Alu DNA in genomic chromatin from nucleases during apoptosis. However, such an interpretation is too restrictive since it does not explain why CpG motifs in Alu sequences in free DNA are hypomethylated, or why Alu and LTR/HERV elements are transcribed in AID patients, nor does it address the fact that Alu RNA can be preferentially reverse transcribed by an active reverse transcriptase [98]. Li and Steinman, with their observation of abundant Alu DNA in SLE sera, suggested reverse transcriptase activity as one possible explanation [101]. This fits well with the polyamine

hypothesis since the proposed decreased SAM levels could lead to progressive hypomethylation of genes, resulting in expression of previously sequestered endogenous reverse transcriptases and Alu elements, particularly the Alu cluster in the PAR1 and the “young” LINE1 elements in Xp. Considering Alu DNA, it has been proposed that Alu DNA reverse transcribed in the cytoplasm could form autoantigenic complexes by binding nascent histones, which are out of their normal epigenetic context when encountered in the cytoplasm, and non-histone proteins, such as the Ku autoantigen which binds Alu DNA [102]. In addition, these Alu DNA fragments, which are very rich in CpG, would be hypomethylated and could be interpreted by the immune system as foreign DNA. Hypomethylated CpG rich DNA in SLE patients is able to activate the immune system via toll-like receptors [103].

Polyamine Hypothesis of AID Defined We can define the polyamine hypothesis as: an abnormal increase in polyamines and polyamine synthesis and recycling leading to problems with cellular methylation; and creation of reactive by-products and abnormal conju-

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gated proteins; stabilization of alternate nucleic acid conformations; altered membrane and ion channel functioning, and interference with localization and assembly of macromolecular complexes. This can result in endogenous material that is autoantigenic [5, 6, 104]. Some possible ways in which the increase in polyamine synthesis and recycling could occur are from induction of the enzymes by a pathogen (e.g. ,EBV inducing c-MYC and ODC) or from loss of epigenetic control of X-linked polyamine genes (e.g., SSAT) due to aging or repeated stresses.

Conclusions This hypothesis gives a comprehensive explanation for many AID autoantigens. The hypothesis begins with cellular stress that stimulates the polyamine pathway, putting increased demand on SAM levels, which can lead to hypomethylation. This hypomethylation can then allow for expression of previously sequestered genes, such as on the Xi. Of interest are Alu elements, HERVs, LINE1 reverse transcriptases, RNA pol III activity, and X-linked polyamine genes, SSAT and SMS, which could lead to wasteful cycling through the polyamine pathway. Reduced SAM levels can also lead to abnormal localization and processing of proteins and RNA. Expression of Alu RNA can disrupt macromolecular complexes (e.g., SRP) and be a source of hypomethylated DNA through reverse transcription. Autoantigens that appear in an individual would be dependent on the cell type in which the mechanism occurs and the extent of disruption in the cell. Polyamines are also elevated in many cancers. Genes in Xp22 are suspected in breast cancers [105, 106]. And so the polyamine pathway has promising therapeutic targets for cancer and AID, but effective treatment will probably require drug combinations targeting multiple enzymes.

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