text:
Epigenetics in Myeloproliferative Neoplasms

Abstract A decade on from the description of JAK2 V617F, the MPNs are circumscribed by an increasingly intricate landscape. There is now evidence that they are likely the result of combined genetic dysregulation, with several mutated genes involved in the regulation of epigenetic mechanisms. Epigenetic changes are not due to a change in the DNA sequence but are reversible modifications that dictate the way in which genes may be expressed (or silenced). Among the epigenetic mechanisms, DNA methylation is probably the best described. Currently known MPN‐associated mutations now include JAK2, MPL, LNK, CBL, CALR, TET2, ASXL1, IDH1, IDH2, IKZF1 and EZH2. Enhancing our knowledge about the mutation profile of patients may allow them to be stratified into risk groups which would aid clinical decision making. Ongoing work will answer whether the use of epigenetic therapies as alterative pathway targets in combination with JAK inhibitors may be more effective than single agent treatment.

Introduction
Genomic instability is fundamental to the development of cancers [1]. The myeloproliferative neoplasms (MPNs) arise when acquired mutations in haematopoietic stem cells cause a change from the polyclonal haematopoiesis seen in health to an abnormal monoclonal haematopoiesis [2]. The classic BCR/ABL-negative MPNs include polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). They are defined, respectively, by the excess production of erythrocytes, platelets and bone marrow fibrosis. The MPN symptom burden is heterogeneous, ranging from patients who are relatively asymptomatic to those suffering with severe constitutional symptoms, thrombotic disease or transformation to secondary acute myeloid leukaemia (AML). Transformation to AML is the most devastating complication experienced by MPN patients, seen in 20% of PMF, 4.5% in PV and 1% in ET [2][3][4][5]. It is usually refractory to treatment and in the majority of cases patients ultimately die of their disease. Therefore, early predictors of leukaemic risk are hugely important [6]. A single-point mutation in the tyrosine kinase JAK2 is present in the majority of patients and has become part of the formal diagnostic criteria for these conditions [7][8][9][10]. However, it is not present in every case and does not explain the phenotypic differences between these three diseases and even between patients. A decade on from the description of JAK2 V617F, the MPNs are circumscribed by an increasingly intricate landscape [11]. The influence of molecular abnormalities beyond the JAK/STAT pathway is ever more being scrutinised. There is now evidence that the MPNs are likely the result of combined genetic dysregulation with several mutated genes involved in the regulation of epigenetic mechanisms. They appear to be the most frequent after JAK2 V617F and CALR mutations. In this review, we explore the evidence for epigenetic dysregulation in the MPNs, where recent advances may be helpful in the need for riskadapted therapeutic stratification.

Mutations affecting cell signalling
Normal haematopoiesis is regulated mainly by cytokines such as erythropoietin (EPO), granulocyte colony-stimulating factor (GCSF) and thrombopoietin (TPO). These cytokines stimulate receptors on the surface of cells and activate the associated Janus kinase (JAK). This protein autophosphorylates and binds to a STAT (signal transducer and activator of transcription) protein, which is in turn itself phosphorylated. The STAT protein then dimerizes and translocates into the nucleus of the cell. Here, it binds to DNA at a promoter site and causes gene transcription ( Fig. 1: JAK/STAT pathway). STAT proteins affect basic cell functions such as cell growth, differentiation and apoptosis.
The point mutation JAK2 V617F was initially described in 2005 [2,[12][13][14][15], located in exon 14 of the gene, and it results in a single base change from guanine to thymine at nucleotide position 1849, leading to an amino acid change from valine to phenylalanine at codon 617. This leads to constitutively active JAK2 kinase signalling that is independent of cytokine stimuli. Approximately 95% PV patients, 50-70% ET patients and 40-50% PMF patients possess the JAK mutation [2,16]. However, initial excitement was short-lived when it became clear that its role in MPNs was not as pathognomonic as BCR/ABL in chronic myeloid leukaemia (CML) [1]. JAK2 V617F positivity or high mutant allele burden does not seem to have a clear association with survival, leukaemia transformation or risk of thrombosis [17]. In recent years, several other mutations in this pathway have been described. A second mutation in JAK2 can occur at exon 12 with similar functional consequences to the canonical mutation [2,18]. It occurs in a small percentage of JAK2 V617F-negative PV patients, but not in ET or PMF. The myeloproliferative leukaemia virus oncogene (MPL) encodes the receptor for TPO via JAK-STAT signalling. Somatic mutations affecting exon 10 of MPL are seen in up to 15% of JAK2 V617F-negative ET and MF patients [2,19,20]. This gain-offunction mutation is due to a substitution of tryptophan to leucine at codon 515 and leads to constitutive phosphorylation of JAK2, STAT3, STAT5, ERK and AKT proteins [21][22][23]. Lymphocyte-specific adaptor protein (LNK) negatively regulates TPO/EPO signalling, and a loss-offunction mutation is found here at low frequency in JAK2-negative patients [24,25]. Further downstream, mutations in suppressors of cytokine signalling (SOCS) and Casitas B cell lymphoma (CBL) have been seen rarely in within MPN patients. In 2013, a mutation in exon 9 of the calreticulin (CALR) gene was identified, by next-generation sequencing (NGS), in approximately 73% of JAK2/MPL-negative ET and MF patients [26,27]. CALR is present in the endoplasmic reticulum, where it forms a key component of the quality-control machinery that ensures proper glycoprotein folding and it also contributes to calcium homeostasis. Recent work has demonstrated the exact mechanism by which CALR causes the MPN phenotype, where the mutant proteins interact with the thrombopoietin receptor (MPL) and directly lead to dimerization and activation of JAK2 [28][29][30]. Retrospective analysis of clinical data suggests that CALR-positive patients have a more benign clinical course than patients with the same MPN who are JAK2 or MPL positive [26]. Compared to JAK2 patients, CALR patients have a lower risk of thrombosis and longer overall survival (OS) [26]. Identification of these additional mutations means that up to 95% of patients can now be reliably diagnosed ( Fig. 2: Frequency of MPN-specific mutations).

Mutations in epigenetic-associated genes
In a study using targeted NGS of 104 cancer-related genes on 197 MPN patients to examine clonal evolution over time [31], approximately 10% of patients had no mutation detectable in any of the 1661 genes analysed and 54% had mutations only in JAK2 V617F or CALR. These patients were found to have the most favourable prognosis and the lowest risk of disease progression. The remaining 36% had additional mutations detected, other than JAK2 V617F or CALR. Most of these were mutations affecting genes implicated in epigenetic regulation (TET2 12%, ASXL1 5%, DNMT3a 5%, EZH2~3% and IDH1 1.5%) [31]. Many mutations in epigenetic-associated genes are not exclusive to MPN and are seen in a wide spectrum of myeloid malignancies. They can also coexist with JAK2/CALR/MPL suggesting that in some cases, these two classes of mutations may combine together produce the individual MPN phenotype. It is thought that the rate of clonal evolution in MPN is slow and most mutations are already present at diagnosis. However, the order in which mutations are acquired may also affect disease phenotype. Of note, the reversible nature of epigenetic changes may make them good potential therapeutic targets.
Epigenetic changes are not due to a change in the DNA sequence but are reversible modifications that dictate the way in which genes may be expressed (or silenced). It can do this through alterations in DNA methylation, modification of histones or chromatin structure, or via changes to RNA.
To understand these epigenetic changes, it is essential to understand the structures that aid the packaging of DNA within the cell nucleus ( Fig. 3: The nucleosome). The nucleosome consists of a core of eight histone proteins (two of histone H2A, H2B, H3 and H4) around which DNA is tightly coiled. This structure is further stabilized by histone H1. DNA is negatively charged, while lysine residues on histones hold a positive charge. The nucleosome is not static and changes in the condensed nature of chromatin signal for changes in gene transcription.

JAK2
In addition to its prominent role in cytokine signalling, mutant JAK2 may have an epigenetic role through its presence in the nucleus where it can lead to phosphorylation of histone H3 and the arginine methyltransferase PRMT5 [32,33] (Fig. 4: The epigenetic effects of JAK2). Phosphorylation of H3 at position Y41 blocks the binding of heterochromatin protein 1a leading to changes in gene transcription, DNA repair and other cellular processes [21]. Phosphorylation of PRMT5 prevents its association with methylosome protein 50 (MEP50), leading to decreased methylation of histones H2A and H4.

DNA methylation
Among the epigenetic mechanisms, DNA methylation is probably the best described. The addition of methyl groups to DNA typically occurs predominately at CpG sites (cytosine and guanine separated by one phosphate in the linear sequence of bases along the length of the DNA) prior to the promoter regions of genes [34,35]. Cytosines are methylated by the addition of a methyl group to the five carbon ring of the pyrimidine ring under the action of DNA methyltransferase (DNMT) enzymes; the 5-methylcytosine (5-mc) formed acts to repress gene transcription. The methyl group (CH 3 ) is donated by Sadenosyl methionine (SAM), which itself is reduced to S-adenosyl homocysteine (SAH). Conversely, the ten-eleven translocation (TET) proteins belong to a family of alpha-oxaloglutarate-dependent enzymes which catalyse the conversion of 5-mc to 5-hydroxymethylcytosine (5-hmc), an initial step in demethylating DNA that ultimately leads back to an upregulation of transcription. Isocitrate dehydrogenase (IDH) enzymes catalyse the conversion of isocitrate to a-ketoglutarate, a reaction that is required for the function of TET enzymes. These mechanisms (Fig. 5: Overview of DNA methylation) help to regulate gene expression in normal development. In the development of cancer, a gain of methylation in a tumour suppressor gene would lead to gene inactivation and a loss of methylation in proto-oncogenic genes would lead to gene expression [36].
Mutations of TET2 impair the function of TET enzymes, resulting in reduced levels of 5-hmc. As discussed above, they are seen in 16% PV, 5% ET and 17% PMF [37]. They also occur in mastocytosis, AML, myelodysplastic syndromes (MDS) and more than half of chronic myelomonocytic leukaemia (CMML) cases [37,38]. Murine models with TET2 mutations had an expansion of the haematopoietic stem cell compartment and in particular demonstrated myelomonocytic proliferation [39]. In one study of MPN patients by Lundberg et al., TET2 mutations were seen to confer a high risk of leukaemic transformation (increased by 30%) and shorter OS [31]. In addition, there is evidence that TET2 mutations appear to be an early event that may provide clonal advantage and set up a 'fertile ground' for MPN disease initiation [40,41].
DNMT 3A and 3B (alpha and beta) carry out de novo methylation while DNTM1 maintains existing methylation patterns after cell division. Mutations in DNMT3A were initially found in AML patients and confer an intermediate to adverse prognosis. They have been seen less frequently in MPN, in approximately 10% of patients [2]. They may cause a gain or loss of function. The most common mutation R882H causes a loss of function and the consequently reduced methylation acts to increase gene transcription with murine models displaying myeloid proliferation with thrombocytosis. Initially, DNMT3A mutations were only reported early in myeloid disease, prior to gaining JAK2 [42]. However, it seems that these mutations can also be acquired late in disease development and the order of acquisition may be associated with differences in MPN phenotype [42]. When they occur prior to JAK2, they are associated with ET, whereas the reverse is associated with PV. However, studies to date have not clearly linked DNMT mutations with clinical outcome in MPN patients.
The enzymes IDH 1 and 2 (IDH1/2) map to chromosomes 2 and 15, respectively. IDH1/2 mutations were first described in 2008 in gliomas and occur at low frequency in MPN. The largest study of IDH mutations in MPN reported frequencies of approximately 2% in PV, 1% ET, 4% PMF and 22% in blast phase MPN [43]. In blast phase, MPN IDH mutational status predicted poor prognosis.
Most DNA methylation studies in MPN have been carried out looking at single-gene mutations, but one study has looked at the epigenetic landscape by examining global DNA methylation in 71 chronic PV/ET/PMF patients [36]. An aberrant methylation pattern was seen in these patients when compared to healthy controls, but no difference was seen between the three MPN phenotypes. However, in 13 of patients samples taken during transformation to secondary AML were also examined and these had an increased number of differentially methylated regions when compared to the samples from chronic patients [36]. Interestingly, analysis of the enriched genes indicated they were involved in important signal transduction pathways such as NFkB (nuclear factor kappa light-chain enhancer of activated B cells) and binding sites for important transcription pathways such as globin transcription factor 1 (GATA1). GATA1 has critical functions in erythrocyte development including establishment of the cytoskeleton and polypeptide globin chains. In patients who transformed to AML, genes in the interferon (IFN) pathway were seen to be hypermethylated, which may be important given IFN has a role in tumour surveillance.

Histone modifications and regulation of chromatin structure
The N terminal lysine residues on histones are prone to post translational modification, leading to transcriptional activation or repression. These covalent modifications can include acetylation, methylation and phosphorylation, among others. Acetylation occurs at lysine (K) residues particularly on histone H3 and H4. Histone acetyl transferases are enzymes that aid the transfer of acetyl groups, while histone deacetylases (HDACs) remove acetyl residues from histone tails. Disruption of this equilibrium can play a role in the development of cancer and have been described in AML. The methylation of histones takes place on lysine, arginine and histidine residues. An additional layer of complexity is added as lysines may be mono-(me), di-(me2) or tri-(me3) methylated. As with DNA, SAM donates the methyl group under the action of histone methyltransferase (HMT) enzymes. Certain commonly studied histone methylation marks are in general associated with transcription activation (H3K4: histone H3 at lysine (K) amino acid 4) while others are associated with transcriptional repression (H3K9).
Yet little is known about the role of individual histone modifications within MPNs, and current research has focused mainly on changes relating to the polycomb group proteins. These are a family of proteins which can remodel chromatin thus exerting a repressive effect on gene expression. Polycomb repressive complex 2 (PRC2) catalyses di-and trimethylation of lysine 27 on histone H3 (H3K27me2/3), leading to gene silencing. Addition of sex combs like protein 1 (ASXL1) maps to chromosome 20 and is thought to be an important mediator of PRC2 function [44]. Mutations in ASXL1 were first described in 2009 in MDS/AML [45]. Studies of the effect of ASXL1 mutations in MDS patients demonstrated that they predicted for worse outcome [46]. In murine models, loss of asxl1 resulted in progressive multilineage cytopenias and dysplasia [47]. Deletions, insertions and nonsense mutations of ASXL1 have been found in 3% ET and 13% PMF [37]. Among patients with MPNs, ASXL1 mutations appear to be commoner in older patients as well as in patients with PMF or post-ET/PV MF. In PMF, they are associated with a more severe anaemia and the inferior survival of patients [48].
EZH2 (enhancer of zeste homolog 2) is another key regulator of PRC2 [49]. EZH2 is present on chromosome 7 and is overexpressed in solid tumours such as lymphoma. MPN-associated EZH2 mutations result in a loss of function and may have tumour suppressor activity [37]. Both EZH2 and ASXL1 are thought to occur later in MPN development, but to date, there is no definite evidence that they carry any independent prognostic value.

miRNA regulation
Changes in the regulation of RNA can lead to cell cycle arrest and apoptosis. Described mutations here include SF3B1, SRSF2 and IKZF1 (IKAROS family zinc finger 1). SF3B1 mutations are seen most often in a subtype of MDS (RARS-refractory anaemia with ring sideroblasts), while SRSF2 is seen in CMML. They can also occur at low frequency in MPN patients (around 5%) with the suggestion that SRSF2 confers poor prognosis in MF. IKZF1 deletions are rare in chronic phase MPN but have been detected at transformation to AML. They have been previously described mainly in lymphoid diseases and are believed to modulate expression of lineage-specific genes [17]. initiate treatment [40]. Current prognostic scoring systems for ET and PV do not take into account any genetic markers and are based solely on patient age, blood counts and development of complications. Standard treatments for the classic BCR/ABL-negative MPNs include lowdose aspirin, cytotoxic drugs such as hydroxycarbamide or anagrelide, venesection and IFN. In younger patients with high-risk MF or transformation to secondary AML, intensive chemotherapy followed by bone marrow transplantation may be considered. In 2011, the JAK1/2 inhibitor ruxolitinib (also known as INCB18424) was approved for the treatment of intermediate-risk and high-risk myelofibrosis. In clinical trials, when it was compared to the best available therapy (BAT), ruxolitinib resulted in a rapid reduction in spleen size (35% or more from baseline by MRI or CT) and also improved constitutional symptoms (including anorexia, shortness of breath, fatigue, insomnia and pain) leading to improved quality of life [50]. In a subsequent 3year follow-up study (COMFORT-II), longer overall survival (OS) was also seen in patients on continuous ruxolitinib [51]. This medication is now being trialled in PV and ET. 'RESPONSE' evaluated the safety and efficacy of ruxolitinib in 110 PV patients, compared to BAT. Ruxolitinib was superior in controlling haematocrit, reducing spleen volume and improving symptoms such as sweats, itch, lethargy and abdominal discomfort [52]. Preliminary reports from the investigator lead study 'MAJIC' suggest that for the treatment of ET, ruxolitinib offered no advantage in terms of complete haematological remission rates, compared to BAT. However, once again benefits were seen for constitutional symptoms with patients reporting improvement in pruritus, weight loss and early satiety [53]. Despite clear clinical benefits, treatment with ruxolitinib does not seem to reduce mutant allele burden, neither in patients nor in mouse models [54]. This is in contrast to the effect of the tyrosine kinase inhibitors on BCR/ABL in CML and has caused researchers to question whether JAK2 is an essential therapeutic target in MPN. The concept of targeted therapy in MPN may be more complicated than first thought. Other JAK inhibitors are in various stages of investigation for the treatment of MF. These include CYT387 which has demonstrated efficacy in reducing spleen size as ruxolitinib did, but with the added benefit of decreasing anaemia [55]. Pacritinib (SB1518), a dual JAK2/Fms like tyrosine kinase 3 inhibitor, has also proved efficacious in reducing spleen size and constitutional symptoms and is being trialled in patients with low baseline platelet counts (PERSIST-1 and PERSIST-2) [56,57]. A major advantage of these trials against the ruxolitinib trials was that patients with any degree of cytopenias were eligible. However, the US Food and Drug Administration (FDA) placed a full clinical hold on pacritinib following reports of patient deaths related to cardiac failure and intracranial haemorrhage. The full clinical data from these studies are under review, and further dose finding studies may have to be performed. Several other agents including fedratinib (SAR302503), CEP-701, XL019, LY278544, BMS-911543 and AZD 1480 have been removed from trials due to various complications including neurological toxicities, myelosuppression or insufficient efficacy [40].

JAK inhibition and potential for combination with epigenetic therapies
A variety of novel small molecule therapies are under investigation in combination with JAK inhibitors. Given the landscape of epigenetic mutations alongside JAK2 mutations, it is possible that drugs such as histone deacetylase inhibitors (HDAC) like Givinostat [58,59], telomerase inhibitors (imetelstat), or DNMT inhibitors could work synergistically with JAK inhibitors. There is no question that hypomethylating agents (azacytidine and decitabine) have offered clinical benefit to selected MDS/AML patients [60]. Other targeted therapies include PI3K (phosphoinositide 3-kinase) inhibitors or mammalian target of rapamycin (mTOR) inhibitors which target enzymes which are part of the PI3K/AKT/mTOR pathway [40]. There are many components within this pathway and inhibition may result in tumour suppression. A trial of a PI3K inhibitor used in combination with ruxolitinib showed antiproliferative effects [61]. Despite recent advances, the true role of molecular status in the choice of treatment of individual patients requires further work before it can be applied routinely [40].

Conclusions
Mutations in MPN are involved in a wide range of cellular pathways including cytokine receptor signalling but also the regulation of gene expression [2]. Currently known MPN-associated mutations include JAK2, MPL, LNK, CBL, CALR, TET2, ASXL1, IDH1, IDH2, IKZF1 and EZH2 (Table 1: Mutations in epigenetic regulators and their current prognostic value in myeloid malignancies) [36]. In other myeloid malignancies, AML and myelodysplastic syndrome (MDS), the number of mutations at diagnosis correlates with time to leukaemic transformation. In these patients, molecularly guided therapeutic trials have led to improved risk stratification and the development of targeted therapy [6]. However, in MPN, certain mutations may be present at disease initiation and others acquired in a random fashion throughout the disease course. In addition, some of these mutations are not mutually exclusive, making the hierarchy complex and unpredictable. This makes it more difficult to predict MPN patient outcomes at a single time-point [6]. More than a decade on from the original description of JAK2 V617F, it seems that contributing the MPN phenotype to constitutive activation of JAK2 is to over simplify the complex molecular interactions that regulate the JAK/STAT pathway. Along with the recent description of the CALR mutation, differences in the epigenetic landscape have been hypothesised to be a key component, with studies showing aberrantly methylated genes in MPN patients particularly at transformation to AML [36]. Other important insights into MPN pathogenesis have appeared with TET2 seeming to be an important driver of leukaemic transformation [62]. Enhancing our knowledge about the mutation profile of patients may allow them to be stratified into risk groups which would aid clinical decision making. Management of MPN patients has now evolved and requires carefully consideration of individual patient disease burden, comorbidities and molecular status. JAK inhibitors have had a meaningful impact as single agent therapy in PMF and now potentially resistant cases of PV and ET. Ongoing work will answer whether the use of epigenetic therapies as alterative pathways in combination with JAK inhibitors may be more effective than single agent treatment.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Differential regulation of synaptic AP-2/clathrin vesicle uncoating in synaptic plasticity

AP-1/σ1B-deficiency causes X-linked intellectual disability. AP-1/σ1B −/− mice have impaired synaptic vesicle recycling, fewer synaptic vesicles and enhanced endosome maturation mediated by AP-1/σ1A. Despite defects in synaptic vesicle recycling synapses contain two times more endocytic AP-2 clathrin-coated vesicles. We demonstrate increased formation of two classes of AP-2/clathrin coated vesicles. One which uncoats readily and a second with a stabilised clathrin coat. Coat stabilisation is mediated by three molecular mechanisms: reduced recruitment of Hsc70 and synaptojanin1 and enhanced μ2/AP-2 phosphorylation and activation. Stabilised AP-2 vesicles are enriched in the structural active zone proteins Git1 and stonin2 and synapses contain more Git1. Endocytosis of the synaptic vesicle exocytosis regulating Munc13 isoforms are differentially effected. Regulation of synaptic protein endocytosis by the differential stability of AP-2/clathrin coats is a novel molecular mechanism of synaptic plasticity.

Results
AP-2 CCV stability. We isolated synaptic CCV following the established sucrose density gradient centrifugation protocol. There was no difference in the fractionation of AP-2 isolated from wt and σ1B −/− synapses on the gradient. In our first semi-quantitative western-blot analysis we had determined an 80% increase in synaptic AP-2 and a 120% increase in synaptic AP-2 CCV 2 . The clathrin basket forms the outer layer of CCV. The clathrin-heavy-chain (CHC) forms triskelia, which connect via their proximal and distal legs building hexagonal and pentagonal lattices. Basket disassembly requires Hsc70, which is recruited to its site of action by its co-chaperones auxilin-1 and −2. However, Hsc70 has additional basket binding sites [11][12][13][14][15][16][17][18][19][20][21] . In our previous analysis we found no increase in Hsc70 in σ1B −/− synapses, but a 100% increase in synaptic CCV associated Hsc70 compared to wt CCV 2 . Thus the majority of AP-2 CCV in σ1B −/− synapses undergo rapid uncoating similar to wt CCV and the increase in AP-2 CCV could be solely due to enhanced CME, despite the reduction in SV recycling. Could the AP-2 CCV half life be extended as well, due to delayed uncoating of the CCV? Activated Hsc70 binds CHC in a dynamic way and mediates basket disassembly by permanent 'collision pressure' against the basket from its inside. If Hsc70 is not guided to the basket uncoating domain, it will bind comparably stably and promiscuously to other CCV protein domains 11 . We reasoned that if a stabilised AP-2 CCV pool exists in σ1B −/− synapses, those CCV should have a different, stable mode of Hsc70 binding, which might allow their separation from the total CCV pool.
For the analysis of CCV stability one has to be aware, that their protein coat is not a rigid protein network. CCV are unstable in physiological buffers. CCV isolation requires slightly acidic pH in order to stabilise their coat. Raising the pH to slightly alkaline pH afterwards is sufficient to induce coat disassembly [22][23][24] . We first isolated synaptic CCV at pH 6.4 on a sucrose gradient following the established protocol 2,25 . Next we performed an anti-Hsc70 CCV immunoisolation from the pooled CCV gradient fractions. Antibodies isolated CCV from σ1B −/− synapses and importantly also from wt synapses. Immunoisolated CCV from σ1B −/− synapses contain two times more AP-2 than the corresponding pool from wt mice, similar to the AP-2 ko/wt ratio found in the total synaptic CCV pool. We also compared the AP-1 content of these CCV from wt and σ1B −/− synapses and found it to be identical (Fig. 1A,B). Thus AP-1 CCV formed in σ1B −/− synapses are not stabilised compared to the corresponding wt CCV. The molecular mechanisms responsible for CCV half life extension have to be specific for AP-2 CCV and this particular endocytic pathway.
Immunoisolated CCV from σ1B −/− synapses have slightly more CHC, but they contain two times more clathrin-light-chains (CLC) (Fig. 1A,B). CLC bind to the proximal legs and take part in the regulation of basket stability and life time 14 . Tissues vary in their CHC/CLC ratios. Only in brain is the ratio 1, while other tissues contain less CLC than CHC. The increase in CLC indicates that these CCV from σ1B −/− synapses are indeed stabilised compared to those from wt synapses. The large GTPase dynamin is essential for the last step in CCV formation. It assembles into rings, constricts the vesicle neck and detaches CCV from the plasma membrane 26,27 . CCV immunoisolated from σ1B −/− contain more dynamin than the respective wt CCV. The total CCV pool of σ1B −/− synapses contains less dynamin than wt CCV, which would be in line with a fast CCV uncoating reaction (Fig. 1A,B). This increase is in line with slow dynamin ring disassembly and thus slow CCV uncoating.
CSPα is a presynapse specific chaperone known to be incorporated into CCV [28][29][30] . Previously we have shown that synaptic CCV from σ1B −/− synapses contain more of it than wt CCV 2 . Also the immunoisolated CCV contain more CSPα than the corresponding wt CCV, but the increase is comparable with the increase in the total σ1B −/− synapse CCV pool 2 (Fig. 1A,B). Thus CSPα does not play a role in CCV stabilisation.
Next we analysed whether immunoisolated CCV from σ1B −/− synapses have less of Hsc70 and auxilin-1/−2 than those CCV from wt synapses. In our previous analysis we detected an increase in Hsc70 in the total synaptic CCV pool which matches the increase in AP-2 2 . However there is no difference in the amount of Hsc70 associated with CCV immunoisolated from wt and σ1B −/− synapses, despite the increase in AP-2 CCV. This reduced AP-2:Hsc70 ratio and reduction in Hsc70 is in line with a stabilised pool of AP-2 CCV (Fig. 1C,D). Surprisingly, more auxilin-1/2 co-chaperones are associated with CCV from σ1B −/− synapses (Fig. 1C,D). Regulatory mechanisms apparently prevent Hsc70 activation by auxilin-1/−2 and Hsc70 binds to CCV sites, at which it is not undergoing ADP-ATP cycles and does not disassemble the clathrin basket 11 .
Firstly, these data demonstrate the existence of a more stable AP-2 CCV pool within a synapse. Secondly, they demonstrate that this AP-2 CCV pool is increased and more stabilised in σ1B −/− than in wt synapses. Next we tested whether also AP-2 membrane binding is stabilised.
Scientific RepoRts | 7: 15781 | DOI:10.1038/s41598-017-16055-4 AP-2 hyperactivation. AP-2 recruits clathrin to the membrane and the CCV life cycle is controlled via the regulation of AP-2 membrane binding. High affinity AP-2 membrane binding requires its binding to cargo proteins and to PI-4,5-P 2 (PIP 2 ). These interactions are regulated and require a conformational change of AP-2. This reorientation releases a steric blockade of the cargo protein binding domains in μ2 and σ2 adaptins and brings the PIP 2 binding motif of μ2 in proximity to the membrane. This transition is supported by μ2-Thr 156 phosphorylation by the AAK1 kinase ( Fig. 2A). AAK1 is not the only kinase phosphorylating μ2, but these kinases have not yet been identified [31][32][33][34][35] . We determined the amount of activated AP-2 complexes using a μ2-Thr 156 -Pi specific antiserum 33,36 . This revealed an increase of μ2-Pi by 35% in the total synaptic CCV pool of σ1B −/− synapses compared to wt CCV in our previous study 2 . This is less than expected given the increase in AP-2, which again suggests a higher turnover rate of the majority of AP-2 CCV in σ1B −/− synapses compared to wt synapses. Also less AAK1 kinase is associated with these CCV compared to wt CCV 2 . In immunoisolated CCV from σ1B −/− the μ2-Pi level is increased by 215% compared to the wt CCV. AAK1 is increased by 100% in these CCV compared to the corresponding wt CCV (Fig. 2B,C). This is a second molecular mechanism for the differential stabilisation of these AP-2 CCV.
Less synaptojanin-1 recruitment. AP-2 membrane dissociation requires PIP 2 phosphatase activities of ubiquitous synaptojanin-1, and of the brain-specific splice variant with a C-terminal truncation 37 . σ1B −/− cortices contain less synaptojanin-1, but synapses contain 20% more than wt synapses, again in line with high AP-2 CCV turnover. The amounts of the brain specific splice variant are however not changed. The total CCV pool of σ1B −/− synapses contains normal amounts of synaptojanin-1, the splice variant is reduced by 20% (Fig. 3A,B). This isoform lacks the AP-2 binding site and is recruited at a late stage of CCV formation via binding to e.g. endophilin and amphiphysin, suggesting isoform specific functions in the regulation of CCV uncoating 37 . However, immunoisolated CCV from σ1B −/− synapses contain 80% less of both synaptojanins than the respective wt CCV (Fig. 3A,B). Mechanisms preventing synaptojanin-1 recruitment to stabilised CCV are not isoform specific. This is a third molecular mechanism of AP-2 CCV stabilisation and thus half life extension.
Synaptojanins are recruited by binding to endophilin A1 37,38 . Endophilin A1 levels are with ~10% slightly increased in cortices and synapses of σ1B −/− mice. The total synaptic CCV pool contains 15% less endophilin A1 compared to wt CCV. However, the stabilised AP-2 CCV from σ1B −/− and wt mice contain the same amount of endophilin A1 (Fig. 3C,E). The amount of endophilin A1 does not determine the amount of synaptojanins recruited to these CCV during their formation. Their interaction appears to be inhibited.
Synaptojanin-1 is also bound by amphiphysin, which is recruited by AP-2. It stimulates dynamin and organizes the actin cytoskeleton [39][40][41][42][43][44][45] . Amphiphysin levels are only slightly increased in cortices and synapses of σ1B −/− mice, but it is reduced in the total synaptic CCV pool and in the immunoisolated CCV by 35% and 45% respectively (Fig. 3D,E). Thus the reduced synaptojanin-1 association is not simply a consequence of less amphiphysin recruitment. Also these data point to altered regulatory modifications.

Co-adaptor composition of stabilised AP-2 CCV.
Stabilised AP-2 CCV should represent a specific synaptic CME route, but they also may represent just an early stage in the CCV uncoating reaction. However, synaptojanin-1 data strongly indicate formation of two different classes of AP-2 CCV and thus two AP-2 endocytic routes.
To further verify that different AP-2 CCV classes and CME pathways exist, we tested for the incorporation of co-adaptor proteins AP180 and NECAP-1. AP180 is a neuron specific co-adaptor. It binds CHC with a higher affinity than AP-2 and binds cargo proteins not bound by AP-2 46 . Previously we have shown that the total synaptic AP-2 CCV pool of σ1B −/− synapses contains 30% less AP180 than wt CCV 2 . Stabilised CCV of σ1B −/− synapses contain however the same amount as the corresponding wt CCV (Fig. 3F,G). NECAP-1 regulates the interaction of AP-2 β2 with CHC by competing with CHC for the same β2 binding site, which contributes to the regulation of CCV coat polymerisation [47][48][49] . We had found NECAP-1 to be increased by 120% in the total synaptic AP-2 CCV pool of σ1B −/− synapses compared to wt CCV, which matches the increase in AP-2 2 . The stabilised AP-2 CCV isolated from σ1B −/− synapses contain however as much NECAP-1 as the wt CCV, despite the increase in AP-2 ( Fig. 3F,G). These data confirm the existence of two CME routes within synapses.
Next we analysed stonin2 incorporation. It contains a μ-homology domain, homologous to the μ2 cargo binding domain. The only stonin2 cargo known to date is synaptotagmin-1, which is also sorted by SV2A/B [50][51][52] . σ1B −/− cortex and synapses contain ~25% and ~10% less stonin2 compared to the wt. Synaptic CCV from σ1B −/− synapses contain 20% less stonin2 than wt CCV 2 . Stabilised CCV from σ1B −/− synapses contain 130% more stonin2 than the corresponding wt CCV (Fig. 4A,D). Also these data confirm that stabilised AP-2 CCV represent a specific CME route. CME and AZ restructuring. If stabilised AP-2 CCV represent a specific CME route, they should be enriched in specific cargo proteins. Stonin2 is associated with Git1 (GPCR kinase-interacting protein 1), a scaffolding protein organising the cytomatrix and a presynaptic AZ structural protein. Both are arranged in circles at the outer rim of AZ. Git1 is essential for SV recycling 53 . Git1 point mutations associated with schizophrenia show defects in PAK3 (p21 protein (Cdc42/Rac)-activated kinase 3) and MAPK (mitogen-activated protein kinase) activation and signaling 54 . More stonin2 in stabilised CCV suggested that it sorts Git1 into these CCV. The total pool of σ1B −/− synaptic CCV contains 30% less Git1 than wt CCV, but stabilised AP-2 CCV contain 120% more Git1 than the corresponding wt CCV (Fig. 4B,D). Thus Git1 is redistributed from AZ into stabilised CCV, apparently mediated by stonin2. Importantly, Git1 is not endocytosed to be degraded. σ1B −/− synapses contain 50% more than wt synapses (Fig. 4B,D). Git1 is a scaffolding protein with several functional domains. It also takes part in the organisation of the cytoskeleton and the increase in synaptic levels may not be exclusively linked to its functions at the AZ 55 . Git1-deficiency leads to impaired SV recycling 53 and thus its increased level in σ1B −/− synapses could support SV recycling, so it may partially compensate the reduction in SV numbers, suppressing the defect caused by σ1B-deficiency.
Git1 has ArfGAP activity. At the AZ its substrate is Arf6 55 . Arf6 regulates multiple vesicular trafficking routes including CME and SV recycling 56,57 and thus we tested for AP-1/σ1B knockout induced changes in Arf6 levels. Arf6 levels are decreased by 25% in knockout synapses, but are increased by 30% in their total and in their stabilised CCV (Fig. 4C,D). This decrease is not limiting Arf6 recruitment to AP-2 CCV and Arf6 appears not to be involved in the differential regulation of the two CME pathways.
To confirm upregulation of AZ dynamics in σ1B −/− synapses we tested for enhanced CME of Munc13 proteins. We choose those because these AZ proteins bind SV and regulate SV exocytosis kinetics. Munc13 isoforms are AZ specific and are anchored to the AZ by different mechanisms 58 . We compared the content of wt and σ1B −/− synaptic CCV in Munc13-1, ubMunc13-2 and bMunc13-2. In most hippocampal synapses SV priming is solely mediated by Munc13-1, whereas ~10% contain bMunc13-2 or ubMunc13-2. σ1B −/− synaptic CCV contain wt levels of Munc13-1. ubMunc13-2 is reduced by 20% and bMunc13-2 is increased by 20% (Fig. 4E,F). Thus AZ restructuring is clearly induced by the σ1B-deficiency. Interestingly, functions of CME in AZ plasticity are AZ type specific. Future analysis of molecular mechanisms of AZ plasticity has to take this specificity into account. If stabilised AP-2 CCV are specialised in the regulation of AZ plasticity, SV proteins should not be enriched in these CCV. We analysed their content by quantitative protein mass spectrometry (iTRAQ) as described 2 . Stabilised CCV from σ1B −/− and wt synapses were separated in individual lanes on SDS-PAGE, lanes were cut in 24 slices, peptides were generated by trypsin and peptides from wt and σ1B −/− samples were coupled to different iTRAQ labels. In a third lane, stabilised CCV from wt and σ1B −/− synapses were mixed 1:1 and coupled to a third iTRAQ label. Peptides from those 3 lanes were mixed and coupled to a fourth iTRAQ label. Labelling with the third and fourth label should give identical numbers and only proteins which fulfilled this criterium were included in the analysis 2 . We only considered proteins with high sequence coverage and detected several SV proteins (Git1 and stonin2 peptides were not detected). The synaptotagmin-1 (sequence coverage (sc) 42.5%) content was identical in wt and σ1B −/− CCV (Fig. 5). Endophilin A1 (sc 32.4%) levels do not differ as in the semiquantitative western-blot analysis (Figs 3C,E, 4G). Syntaxin-1A and -1B, and SNAP25 (sc 20, 24.3 and 29.1%) were present at comparable amounts in CCV from both genetic backgrounds, but there was less VAMP-2/synaptobrevin-2 (sc 28.4%) in the stabilised CCV from σ1B −/− synapses compared to wt CCV. αand β-synucleins (sc 64.3%, 31.6%) were also present and stabilised CCV from σ1B −/− synapses contained less of them. Stabilised CCV from σ1B −/− synapses also contain less V-ATPase (sc from 3.4% of VATd1, to 63.4%, of VATB2) than the corresponding wt CCV (Fig. 4G). The lower levels in SV proteins confirm the specialised function of stabilised AP-2 CCV in AZ plasticity (Fig. 5).

Discussion
AP-1/σ1B deficiency causes severe X-linked mental retardation. Our analysis of the deficiency induced alterations in pre-synaptic protein traffic reveal novel molecular mechanisms of synaptic plasticity. In previous studies we have shown that this deficiency leads to changes in AP-1 dependent synaptic protein sorting and also induces changes in AP-2/clathrin mediated synaptic endocytosis. AP-1 mediated SV recycling is impaired leading to fewer SV, the concomitant enlargement of early endosomes and their enhanced maturation into multivesicular body endosomes. Enhanced maturation is mediated by endosomal AP-1/σ1A. This AP-1/σ1A function is inhibited by AP-1/σ1B. Alterations in SV dynamics and endosomal protein sorting are accompanied with an increase in AP-2 CCV numbers 1-3 . The increase in AP-2 CCV is a tissue and synapse specific secondary phenotype of AP-1/σ1B-deficiency. Regulation of CME is thus a mechanism of synaptic plasticity 4,6 . We describe the biochemical characterisation of the AP-1/σ1B knock-out induced synaptic CCV. Comparing their coat composition with those isolated from wt synapses revealed that their increase is due to two mechanisms: general upregulation of CME and the stabilisation of a class of these AP-2 CCV, creating AP2 CCV with an extended half life.
We demonstrate three distinct molecular mechanisms, which stabilise the CCV protein coat. In order to identify a function of stabilised AP-2 CCV we analysed their cargo protein content. They are enriched in two major AZ structural proteins, Git1 and stonin2, which assemble into rings at the rim of AZ. Stonin2 is also an AP-2 CCV co-adaptor protein, which binds Git1 53,59 and thus may recruit Git1 into stabilised CCV. Enhanced Git1 CME by these CCV does not lead to its enhanced degradation, synaptic Git1 levels are increased demonstrating Git1 redistribution within σ1B −/− synapses. Upregulation of AZ plasticity is confirmed by the differentially altered CME of Munc13 isoforms, which regulate SV exocytosis. Thus AZ plasticity is regulated via AP-2 CCV mediated endocytosis. In contrast SV proteins are not enriched in stabilised AP-2 CCV and are preferentially sorted by the common CME pathway. This is the first demonstration of a differential regulation of AP-2 CME pathways within a cell.
We identified three distinct molecular mechanisms, which stabilise AP-2 CCV. This is the stable binding of the uncoating ATPase Hsc70, which enabled us to isolate them with anti-Hsc70 antibodies. Stable CCV from σ1B −/− synapses contain more of the Hsc70 co-chaperones auxilin1/2, but they are not able to activate Hsc70. The second mechanism is the hyperactivation of AP-2 by the enhanced recruitment of the μ2 kinase AAK1 and enhanced μ2 phosphorylation. μ2 phosphorylation stabilises AP-2 binding to cargo proteins and to PIP 2 . Both interactions lock AP-2 onto membranes. The third mechanism is the reduced recruitment of the two isoforms of the PIP 2 phosphatase synaptojanin-1. Its activity is essential for membrane dissociation of AP-2.
Next, we have to answer the question about the molecular mechanisms controlling the differential assembly of AP-2 CCV and their half life. The clathrin-basket uncoating machinery is constitutively active, because clathrin readily self assembles in the cytoplasm without the aid of adaptor proteins building empty clathrin cages. If Hsc70 and auxilin-1/2 do not disassemble them continuously, clathrin becomes limiting for CCV formation and CME is blocked 60 . Thus during CME clathrin disassembly by Hsc70 has to be inhibited and has to be activated only after CCV have been formed. In fact, the coat of isolated CCV is unstable in physiological buffer conditions and CCV isolation requires a slightly acidic pH 23 . Cochaperone recruitment to stabilised CCV is not impaired. Interactions of auxilin-1/2 with Hsc70 are prevented, either by post-translational modifications of them or by competitive interactions of auxilins with other coat proteins. The μ2 kinase AAK1 might also regulate Hsc70 function, but our preliminary data also show alterations in additional kinases, which have not been characterised as CCV kinases previously. We have to identify their substrates to demonstrate that their functions indeed regulate CCV stability. They might also modify cargo proteins to direct them into CME. Synaptojanins can be recruited by endophilin A1, but endophilin A1 levels are not reduced in stabilised CCV. Synaptojanins bind to additional CCV coat proteins and thus the regulation of several protein-protein interactions might play a role in their reduced recruitment 37,38,61 .
The CCV immunoisolation procedure and the natural instability of CCV does not allow the precise determination of the ratio of rapid uncoating and stabilised AP-2 CCV, but their pool size can be estimated. The increases in AP-2 in total and stabilised CCV from σ1B −/− synapses are almost identical and thus their μ2-Pi ratios enable us to estimate pool size. Stabilised CCV have 215% more μ2-Pi whereas the total synaptic CCV pool has 30% more μ2-Pi than the corresponding wt pools. We can assume that the 30% increase in the total CCV pool is largely due to the increase in the stabilised pool, because the total CCV pool contains less AAK1, whereas stabilised CCV contain more AAK1. Given the 215/30 ratio, the stabilised pool represents ~15% of the total synaptic AP-2 CCV in σ1B −/− synapses. This small pool size is in line with a specialisation of these AP-2 CCV for the transport of a small subset of synaptic proteins, like the AZ proteins Git1 and stonin2.
The enrichment of AZ rim proteins in stabilised AP-2 CCV suggests that AP-2 CCV formation at the AZ rim is differentially regulated. AAK1 is not the only kinase able to phosphorylate μ2 62 . Our data indicate that a major function of AAK1 is the stabilisation of AP-2 membrane binding and thus the regulation of CCV half life. Other μ2 kinases might regulate the initial activation of AP-2 at specific plasma membrane domains. That AP-complex activation by different kinases is important for the regulation of their protein sorting functions is also indicated by the biochemistry of the AP-1 family. Differential AP-1/μ1A activation could play a role in the differential activation and thus different functions of γ2 and γ1 AP-1 complexes. In addition, the three μ1 isoforms are expected to be activated by different kinases 6,63,64 . Differential AP-2 activation could be achieved by specific targeting of kinases to the site of CME, e.g. by linker proteins or receptor kinase activities, and/or by local kinase activation through second messengers like Ca 2+ , cAMP, DAG etc. Regulation by these second messengers is an attractive model in the case of AZ. In the "shunting" model, high Ca 2+ concentrations ([Ca 2+ ]) at the AZ stimulate SV fusion and inhibit CME at the same time 65 . In the vicinity of AZ, [Ca 2+ ] decreases due to diffusion and CME can take place. SV proteins have to be removed by CME to clear the AZ and to allow repeated SV fusions 8,66 . The low [Ca 2+ ] affinity CME inhibitor has not been identified. Dynamin is inhibited at high [Ca 2+ ], but it is only essential for membrane fission, the final step in CCV formation. However, the phosphorylation levels of CME proteins like PACSIN and intersectin increase upon stimulation of SV exocytosis with [Ca 2+ ] and even more proteins are dephosphorylated 67 . Thus there is no single on/off kinase/phosphatase pair responsible for the coupling of SV exocytosis and CME. Additional regulatory mechanisms could involve the cargo proteins or their specific CCV adaptor protein. Git1 is a scaffolding protein and thus could play the role of a CCV kinome regulator 55 . Stonin2 could be involved as well. Differences in the contents of the AP-2 CCV regulator NECAP-1, but also in the membrane curvature sensing BAR-domain protein amphiphysin and the two synaptojanins, suggest that different CME routes also differ in their CCV budding kinetics, which we will study in the future.
What could be the function of CCV half life regulation and specifically its prolongation? A prolonged CCV half life will delay the fusion of these endocytic vesicles with their acceptor compartment, normally endosomes. A longer CCV half life will also allow their transport over longer distances. Coat proteins do interact with the actin cytoskeleton and AP-2 can function as a linker between cargo and motor proteins 68 . This would allow the fusion of these endocytic vesicles with organelles positioned further away from synaptic plasma membrane, e.g. late endosomes. Late endosomes are involved in slow plasma membrane protein recycling or in receptor signalling pathways. Another reason could be the upregulation of endolysosomal protein degradation in σ1B −/− synapses. Early endosomes mature into late, multivesicular body endolysosomes, which enables protein degradation. Maturation is stimulated by AP-1/σ1A and inhibited by AP-1/σ1B and in σ1B −/− synapses SV protein degradation is upregulated 2,3 . Stabilisation of AP-2 CCV allows their transport past these early endosomes and would prevent delivery of those cargo proteins in the degradative pathway. These cargo proteins can then fulfil functions somewhere else within the synapse. It would also enable their comparatively slow recycling to the plasma membrane and AZ, when required. The stabilisation of AP-2 CCV transporting Git1 indeed appears to prevent its degradation, because synaptic Git1 levels are increased in σ1B −/− synapses. However, it could also be that stabilised AP-2 CCV are not transported away from the plasma membrane and are retained in the vicinity of AZ. After their uncoating the vesicles can fuse back directly with the plasma membrane at the rim of AZ, where Git1 and stonin2 would be repositioned or they would contribute to the formation of a new AZ 53,59 . The increase in synaptic Git1 levels may be able to suppress defects in AP-1 dependent SV protein recycling, because its deficiency leads to impaired SV recycling 53 . How the various domains of Git1 effect AZ functions and SV recycling is not known and thus it is too early to develop a more detailed model about the function of Git1 containing, stabilised AP-2 CCV in AZ plasticity.
This study raises several questions, which will have to be answered in the future. How does stonin2 and Git1 sorting affect AZ function and how are these mechanisms linked to synaptic signalling? How are these AP-2 functions linked to AP-1 functions in SV protein recycling and protein degradation? Currently, we are studying the molecular mechanisms regulating the formation of long lived AP-2 CCV in synapses.

Materials and Methods
Isolation of synaptic CCV. The 'knock-out' mice have been described 1 and also the isolation of synaptosomes and synaptic CCV has been described 2 , which followed established protocols 25 . Isolations from wt and σ1B −/− cortices were always performed in parallel. Animals used for tissue isolations were −/− animals and isogenic + / + animals derived from + /− matings. Animals are kept in the central animal facility of the Faculty of Medicine of the Georg-August-University Göttingen in accordance with the appropriate guidelines. Animals were killed with CO 2 and cervical dislocation in accordance with the appropriate guidelines. Animal housing and the protocol for killing the animals were approved by the,Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit' (LAVES). Brains were isolated from 4-6 month old animals in the afternoon, were snap frozen in liquid nitrogen and stored at −80 °C. Sucrose density gradient fractions containing the purified synaptic CCV were pooled, and incubated with protein G Sepharose bead slurry (Protein G Sepharose 4 Fast Flow GE Healthcare, Uppsala, S) at 4 °C for 1 hour. Beads were pelleted at 2000 rpm and the supernatant was incubated with 5 μg of anti-Hsc70 mouse monoclonal antibody (SYSY, Göttingen, Ger) over night at 4 °C. Protein G Sepharose beads were added at 4 °C for 4 hours. The harvested beads were washed twice with CCV buffer and resuspended in 40μL 3x SDS-PAGE loading buffer. The protein content of the beads and the wash fractions elution 1 and elution 2 were analysed by semi-quantitative western-blot analyses and protein mass spectrometry.
CCV coat protein quantification. Brain extracts were prepared from wt and 'ko' mice in parallel and comparisons of protein content were only made between extracts prepared in parallel. Comparing the data from different animals and independent preparations requires a normalisation and thus wt values were defined as 100%. The protein load was varied between 10-80 μg per lane to determine the linear protein/chemiluminescence signal ratio. Several proteins of different molecular masses were detected on one western-blot nitrocellulose membrane. This served as internal control for protein isolation and detection by the ECL luminescence detection kit PICO (Pierce-ThermoScientific, Karlsruhe, Ger), recorded with a Fuji LAS 1000 (Fujifilm Corp., Düsseldorf, Ger) camera system. Statistics of the quantifications are presented as Bar-plot diagrams, which were generated using DataGraph (Visual Data Tools, USA). Protein determination: Bradford-assay (BioRad, Munich, Ger). Antibodies Quantitative protein mass spectrometry. Proteins separated by SDS-PAGE were subjected to an automated workflow comprising in-gel digestion and subsequent labelling of the tryptic peptides with iTRAQ reagents as already published and described in great detail 2,69 . Labeled peptides were separated by nano-flow liquid chromatography, spotted onto AnchorChip MALDI targets, and analysed on an Ultraflextreme MALDI-TOF/ TOF mass spectrometer (Bruker, Bremen, Ger). Signals were normalised to the control (wt + 'ko') and signal ratios of each sample were normalised to the average deviation of the sample to correct for variations. Values within the standard deviation (variance) ± σ were considered insignificant.


extfieldsofstudy: Biology Medicine

text:
First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum

Histone crotonylation is a new lysine acylation type of post-translational modification (PTM) enriched at active gene promoters and potential enhancers in yeast and mammalian cells. However, lysine crotonylation in nonhistone proteins and plant cells has not yet been studied. In the present study, we performed a global crotonylation proteome analysis of Nicotiana tabacum (tobacco) using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. A total of 2044 lysine modification sites distributed on 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom. Similar to lysine acetylation and succinylation in plants, lysine crotonylation was related to multiple metabolism pathways, such as carbon metabolism, the citrate cycle, glycolysis, and the biosynthesis of amino acids. Importantly, 72 proteins participated in multiple processes of photosynthesis, and most of the enzymes involved in chlorophyll synthesis were modified through crotonylation. Numerous crotonylated proteins were implicated in the biosynthesis, folding, and degradation of proteins through the ubiquitin-proteasome system. Several crotonylated proteins related to chromatin organization are also discussed here. These data represent the first report of a global crotonylation proteome and provide a promising starting point for further functional research of crotonylation in nonhistone proteins.

Results
Detection of lysine-crotonylated proteins in tobacco leaves. To characterize the global crotonylation proteome of tobacco, a proteomic method based on sensitive immune-affinity purification and high-resolution LC-MS/MS was applied to identify crotonylated proteins and their modification sites in tobacco. An overview of the experimental procedures is shown in Fig. 1a. A total of 2044 lysine crotonylation sites distributed in 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom (Table 1). MS/MS information related to these crotonylated peptides were deposited to iProX database with accession number IPX0000889000 (http://www.iprox.org). Detailed information for all identified crotonylated peptides and their corresponding proteins was shown in Supplementary Table S1, the scores for protein and peptide identification were shown in Supplementary Table S2. Among the 637 crotonylated proteins, 357 (56%) proteins contained one or two crotonylation sites, and 80 (13%) proteins had 7 or more crotonylation sites (Fig. 1b). Most peptides ranged from 7 to 28 amino acids in length, consistent with the properties of tryptic peptides (Fig. 1c). To confirm the validation of the MS data, the mass error of all identified peptides was assessed. The distribution of the mass error was near zero, and most of these proteins were less than 0.02 Da, suggesting that the mass accuracy of the MS data met the requirement (Fig. 1d).

Motifs and secondary structures of lysine crotonylated peptides.
To evaluate the nature of the crotonylated lysines in tobacco, the sequence motifs in all identified crotonylated peptides were investigated using the Motif-X programme. As shown in Supplementary Table S3, a total of nine conserved motifs were retrieved. Particularly, motifs KcrE, EKcr and KcrD (Kcr indicates the crotonylated lysine) were strikingly conserved (Fig. 2a, Supplementary Table S4). Importantly, the significantly conserved amino acids in these motifs, namely E and D, were both negatively charged, which were rarely identified in other PTMs. These motifs are likely to represent a feature of crotonylation in tobacco. Hierarchical cluster analysis was also performed to further analyse these motifs. As shown in the heat map (Fig. 2b), the enrichment of positively charged K residues was observed in the −10 to −5 and +10 to +5 positions, while negatively charged residues D and E were markedly enriched in the −4 to +4 position. Short aliphatic A residues were frequently observed in the −10 to +10 position, while the sulphur-containing C residue was not observed.
To explore the relationship between lysine crotonylation and protein secondary structures, a structural analysis of all crotonylated proteins was performed using the algorithm NetSurfP. As shown in Fig. 2c, approximately 47% of the crotonylated sites were located in α-helices, and 12% of the sites were located in β-strands. The remaining 42% of the crotonylated sites were located in disordered coils. However, considering the similarity of the distribution pattern between crotonylated lysines and all lysines, there was no tendency towards lysine crotonylation in tobacco. The surface accessibility of the crotonylated lysine sites was also evaluated. The results showed that 91% of the crotonylated lysine sites were exposed to the protein surface, close to that of all lysine residues (Fig. 2d). Therefore, lysine crotonylation likely does not affect the surface properties of modified proteins.

Functional annotation and subcellular localization of crotonylated proteins.
To obtain an overview of the crotonylated proteins in tobacco, the Gene Ontology (GO) functional classification of all crotonylated proteins based on their biological processes, molecular functions and subcellular locations was investigated (Supplementary Table S5, Supplementary Table S6). Within the biological processes category, the majority of crotonylated proteins were related to metabolic processes, cellular processes, and single-organism processes, respectively accounting for 36, 27 and 24% of all the crotonylated proteins (Fig. 3a). For the molecular function category, 45 and 40% of the crotonylated proteins were associated with catalytic activity and binding functions, respectively ( Fig. 3b). Subcellular localization analysis revealed that most of the crotonylated proteins were localized to the chloroplast (37%), cytosol (30%), nucleus (12%), and mitochondria (5%) (Fig. 3c).

Functional enrichment analysis.
To better understand the biological function of these crotonylated proteins, we performed an enrichment analysis of the GO (Supplementary Table S7 Table S9). The enrichment analysis of the cellular components revealed that the crotonylated proteins were significantly enriched in the proteasome complex, thylakoid membrane, and photosystem II oxygen evolving complex (Fig. 4a). Based on the enrichment results of the molecular function category, most crotonylated proteins were related to NAD binding, threonine-type peptidase activity, endopeptidase activity, and calcium ion binding (Fig. 4a). In the biological processes category, most of the crotonylated proteins were implicated in oxoacid metabolic processes, protein catabolic processes, cellular amino acid metabolic processes, protein folding, ubiquitin-dependent protein catabolic processes, and photosynthesis (Fig. 4a). The KEGG pathway enrichment   analysis showed that a majority of the crotonylated proteins were related to carbon metabolism, carbon fixation in photosynthetic organisms, pyruvate metabolism, proteasome, amino acid biosynthesis, the citrate cycle, glycolysis, porphyrin and chlorophyll metabolism, and photosynthesis ( Fig. 4b). Consistent with these observations, Pfam domains, including the NAD(P)-binding domain, ATPase core domain, chlorophyll a/b binding protein domain, aldolase-type TIM barrel, and thioredoxin domain, were significantly enriched in crotonylated proteins ( Fig. 4c), implying an important role for lysine crotonylation in these processes.

Protein interaction network of the crotonylated proteins in tobacco.
To further identify the cellular processes regulated through crotonylation in tobacco, the crotonylated protein interaction network was established using an algorithm in Cytoscape software. A total of 264 acetylated proteins were mapped to the protein interaction database (Supplementary Table S10), presenting a global view of the diverse cellular functions of crotonylated proteins in tobacco. As shown in Fig. 5, crotonylated protein involved in ribosome, proteasome, carbon metabolism, oxidative phosphorylation, and terpenoid backbone biosynthesis were retrieved, comprising a dense protein interaction network. The physiological interactions among these crotonylated protein complexes likely contribute to their cooperation and coordination in tobacco.

Discussion
Histone crotonylation is a new lysine acylation type of PTM enriched at active gene promoters and potential enhancers in mammalian cells 25 . Crotonylation is catalysed through histone acetyltransferase p300/CBP 28 , 'read' The numbers in X axes represent the value of significant analysis. When the value is greater than 1.3, the p value is less than 0.05, which means the data is statistically significant.
by YEATS2 and AF9, 'erased' by Sirtuin family members SIRT1-3 in yeast and mammals 29,[43][44][45][46] . However, the lysine crotonylation of nonhistone proteins and in plant cells has not yet been studied. To determine whether lysine crotonylation also exists in plants and to study its function in cellular processes, a global crotonylation tobacco proteome was realized using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. A total of 2044 lysine crotonylation sites distributed in 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom. These crotonylated proteins were associated with diverse biological processes, including multiple metabolic pathways, chromatin organization, protein biosynthesis, folding, and degradation. The protein interaction network analysis also suggested that a wide range of interactions involved in these biological processes was likely modulated through protein crotonylation. Carbon is one of the most important macroelements, providing the backbone for biological macromolecules. Lysine acetylation and succinylation in plants have been implicated in carbon metabolism, glycolysis, pyruvate metabolism, TCA cycle, pentose phosphate pathway, glyoxylate and dicarboxylate metabolism 32,33,37,40 . The results of the present study showed that numerous enzymes in these metabolism pathways were also modified through crotonylation. In plants, one of the most important metabolic processes is photosynthesis. In the present study, there are 236 crotonylated proteins were localized to the chloroplast. Among these proteins, a total of 72 proteins were involved in photosynthesis processes. For example, 10, 14, 4, 8, 2, 9, and 25 proteins, identified as members of antenna proteins, photosystems II complex, cytochrome b6f complex, photosystems I complex, ferredoxin-NADP reductase, ATP synthesis complex, and the carbon fixation pathway, respectively. Significantly, 73% (8/11) enzymes in the Calvin cycle 42 were extensively crotonylated at multiple sites, with an average of 10.   Table 3. Crotonylated proteins involved in protein biosynthesis, folding, Ubiquitin-dependent degradation.
For example, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key carbon fixation enzyme, was crotonylated at 15 amino acid sites. The key amino acid residues of Rubisco, K201 and K334 which were identified as acetylated resulting in the downregulation of Rubisco activity 47 , also modified through crotonylation. This result suggested that crotonylation might change Rubisco activity in coordination with acetylation. Moreover, the two Rubisco activase isoforms 48 , involved in the light activation of Rubisco, were also crotonylated at 24 sites. Moreover, 67% (10/15) of the enzymes involved in chlorophyll synthesis 49 were also modified through crotonylation. To our knowledge, until recently, there have been no reports of lysine acylation in chlorophyll metabolism. These results suggested that lysine crotonylation might play a role in regulating carbon metabolism and photosynthesis. Proteins are macromolecules that, in addition to carbohydrates, perform a vast array of functions within organisms. Proteins comprise amino acids and are synthesized through translation. In plants, proteins can be degraded in two ways -proteolysis in the vacuole or via the ubiquitin-proteasome system. The data in the present study revealed that lysine crotonylation was related to the synthesis and degradation of multiple amino acids, such as lysine, valine, leucine and isoleucine. The ribosome serves as the factory of protein synthesis. In the present study, we identified 47 crotonylated proteins associated with translation, including 29 ribosome subunits, 8 translation initiation factors, 7 elongation factors, and 3 aminoacyl-tRNA synthetases. After synthesis in the ribosome, the polypeptide chain rapidly folds into its characteristic and functional three-dimensional structure from a random coil. This process is accomplished through the assistance of chaperones, such as the ER-resident molecular chaperone BiP, the HSP70 family, and the HSP90 family [50][51][52][53][54][55] . The data in the present study showed that lysine residues in members of HSP70 and HSP90 were extensively crotonylated in tobacco. Moreover, Bip 4 and Bip 5 were also extensively modified through crotonylation, suggesting an important role for lysine crotonylation in protein folding. If several rounds of chaperone-assisted folding are futile, unfolded or misfolded proteins are recognized and targeted by ubiquitin and subsequently degraded by proteasomes 56,57 . In the present study, we found ubiquitin, ubiquitin extension protein, ubiquitin-conjugating enzyme, and ubiquitin-activating enzyme, are all modified through crotonylation. Furthermore, 14 proteasome subunits were also crotonylated. These results indicated the likely involvement of lysine crotonylation in regulating protein synthesis, folding, and ubiquitin-dependent degradation.
The organization of the eukaryotic genome into nucleosomes dramatically impacts the regulation of gene expression. The structure of the nucleosome core is relatively invariant in eukaryotic organisms, and includes a 147-bp segment of DNA and two copies of each of the four core histone proteins 58 . Histone chaperone nucleosome assembly protein 1 (Nap1) has been implicated in nucleosome assembly by eliminating competing, nonnucleosomal histone-DNA interactions 59 . The data presented here showed that tobacco histones H1, H2A, H2B, H3, and H4, and nucleosome assembly proteins Nap1;2, Nap1;3, and Nap1;4, were modified through crotonylation, indicating a potential role for lysine crotonylation in nucleosome assembly or disassembly. As complementary evidence, topoisomerase I, required for efficient nucleosome disassembly at gene promoter regions 60 , was also crotonylated in the present study. Nucleosomes are folded through a series of higher-order structures to eventually form a chromosome. An important factor in higher-order organization is the nuclear matrix, which serves as a scaffold for loops of chromatin 61 . Nuclear matrix has been proposed to play a role in regulating transcription, DNA replication, and RNA processing 62 . Chromosomal DNA was anchored to nuclear matrix by its matrix-associated regions (MARs), bound by matrix attachment region-binding protein 63 . Histone acetyltransferase (HAT) p300 and deacetylase SIRT1 interacts with matrix attachment region-binding protein SAF-A and SATB1, respectively, and thereby regulates gene expression 64,65 . Surprisingly, in the present study, a matrix attachment region binding filament-like protein (MFP1) was identified as crotonylated at 20 amino acid sites, and even its homologue was also crotonylated at 8 amino acid sites. MFP1 is a conserved nuclear and chloroplast DNA-binding protein in plants; however, its physiological function is not understood [66][67][68] . Considering that p300 and SIRT1 possess crotonylation and decrotonylation activities, respectively, in animals 25,28,29 , it is an interesting assumption that the crotonylated or decrotonylated form of MFP1 was also associated with the regulation of gene expression. In addition to these crotonylated protein that might be associated with the assembly of nucleosome and chromatin, we identified a G-strand-specific single-stranded telomere-binding protein (GTBP), associated with maintaining telomere stability, also modified through crotonyl groups 69,70 . These results indicated the likely involvement of lysine crotonylation in chromatin organization and gene regulation at least in tobacco.
In summary, the present study provided the first global lysine crotonylation proteome in tobacco. These data revealed lots of crotonylated proteins associated with diverse aspects of cellular process, particularly carbon metabolism, photosynthesis, protein biosynthesis, folding, degradation, and chromatin organization. These finding raised some questions that if the crotonylation of these proteins are related to biological functions and that if crotonylation changes in different situations. All these questions should be addressed in the future work. Nevertheless, the results presented here may provide a promising starting point for further functional research of crotonylation in nonhistone proteins.

Materials and Methods
Plant materials and growth conditions. Tobacco were grown in a greenhouse at 25 °C and a photoperiod of 16/8 h (light/dark). The leaves were excised from 4-week-old seedlings with three biological replicates and immediately used for protein extraction.
Protein Extraction. The samples were grinded to powder in liquid nitrogen, and subsequently mixed with extraction buffer (8 M urea, 2 mM EDTA, 3 μM TSA, 50 mM NAM, 10 mM DTT and 1% Protease Inhibitor Cocktail, Millipore). The remaining debris was removed through centrifugation at 20,000 g for 10 min at 4 °C. Finally, the proteins were precipitated using cold 15% TCA for 2 h at −20 °C. After centrifugation at 4 °C for 10 min, the supernatant was discarded. The remaining precipitate was washed three times with cold acetone. The protein was redissolved in buffer (8 M urea, 100 mM NH 4 CO 3 , pH 8.0) and the protein concentration was determined using the 2-D Quant kit (GE Healthcare) according to the manufacturer's instructions.
Trypsin Digestion. For digestion, the protein solution was reduced with 10 mM DTT for 1 h at 37 °C and alkylated with 20 mM IAA for 45 min at room temperature in darkness. For trypsin digestion, the protein sample was diluted after adding 100 mM NH 4 CO 3 to a urea concentration of less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4-h digestion.
HPLC Fractionation. The sample was subsequently fractionated through high pH reverse-phase HPLC using an Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, the peptides were separated into 80 fractions using a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate, pH 10, over 80 min. Subsequently, the peptides were combined into 6 fractions and dried using vacuum centrifugation. Affinity Enrichment. To enrich Kcro peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, and 0.5% NP-40, pH 8.0) were incubated with pre-washed antibody beads (PTM Biolabs) at 4 °C overnight with gentle shaking. The beads were washed four times with NETN buffer and twice with ddH 2 O. The bound peptides were eluted from the beads using 0.1% TFA. The eluted fractions were combined and vacuum-dried. The resulting peptides were cleaned with C18 ZipTips (Millipore) according to the manufacturer's instructions, followed by LC-MS/MS analysis.
Quantitative Proteomic Analysis by LC-MS/MS. The peptides were dissolved in 0.1% FA and directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient comprised an increase from 6% to 22% solvent B (0.1% FA in 98% ACN) for 24 min, 22% to 40% for 8 min and climbing to 80% in 5 min, subsequently holding at 80% for the last 3 min, all at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system, the resulting peptides were analysed using the Q Exactive TM Plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive TM plus (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. The peptides were selected for MS/MS using NCE setting as 30; ion fragments were detected using Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 5E3 in the MS survey scan with 15.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the Orbitrap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z. Database Search. The resulting MS/MS data was processed using MaxQuant with integrated Andromeda search engine (v.1.5.1.8). Tandem mass spectra were searched against UniProt tobacco database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages, 5 modifications per peptide and 5 charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, crotonylation on Lys and crotonylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for protein, peptide and modification sites were specified at 1%. Minimum peptide length was set at 7. All the other parameters in MaxQuant were set to default values. The site localization probability was set as >0.75.

Bioinformatics Methods.
Motif-X software (http://motif-x.med.harvard.edu/) was used to analyse the model of sequences constituted with amino acids in specific positions of acetyl-21-mers (10 amino acids upstream and downstream of the site) in all protein sequences 71 . For further hierarchical clustering based on categories, all the acetylation substance categories obtained after enrichment were first collated along with their p-values, and subsequently filtered for those categories at least enriched in one of the clusters with a p-value < 0.05. This filtered p-value matrix was transformed by the function x = −log (p-value), and the x values for each category were z-transformed. These z scores were subsequently clustered using one-way hierarchical clustering (Euclidean distance, average linkage clustering) in the Genesis programme. The cluster membership was visualized using a heat map through the "heatmap.2" function in the "gplot2" R-package. Secondary structures were predicted using NetSurfP. Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http:// www.ebi.ac.uk/GOA/). The proteins were classified using Gene Ontology annotation based on three categories: biological process, cellular component and molecular function. The protein subcellular localization was analysed using Wolfpsort (http://www.genscript.com/wolf-psort.html). The KEGG was used to annotate protein pathways. GO term, protein domain, and KEGG pathway enrichment were performed using the DAVID bioinformatics resources 6.7. Fisher's exact test was used to examine the enrichment or depletion (two-tailed test) of specific annotation terms among members of resulting protein clusters. Correction for multiple hypothesis testing was performed using standard false discovery rate control methods. Any terms with adjusted p-values below 0.05 in any of the clusters were treated as significant. The Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database (http://string-db.org/) was used for PPI analysis. Cytoscape (version 3.0) software was used to display the network 72 .