Acetoxymethyl Ester of Tetrabromobenzimidazole-Peptoid Conjugate for Inhibition of Protein Kinase CK2 in Living Cells.

CK2 is a ubiquitous serine/threonine protein kinase, which has the potential to catalyze the generation of a large proportion of the human phosphoproteome. Due to its role in numerous cellular functions and general anti-apoptotic activity, CK2 is an important target of research with therapeutic potential. This emphasizes the need for cell-permeable highly potent and selective inhibitors and photoluminescence probes of CK2 for investigating the protein phosphorylation networks in living cells. Previously, we had developed bisubstrate inhibitors for CK2 (CK2-targeted ARCs) that showed remarkable affinity (KD < 1 nM) and selectivity, but lacked proteolytic stability and plasma membrane permeability. In this report, the structures of CK2-targeted ARCs were modified for the application in live cells. Based on structure-activity studies, proteolytically stable achiral oligoanionic peptoid conjugates of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were constructed. Affinity of the conjugates toward CK2 reached subnanomolar range. Acetoxymethyl (AM) prodrug strategy was applied for loading TBBz-peptoid conjugates into living cells. The uptake of inhibitors was visualized by live cell imaging and the reduction of the phosphorylation levels of two CK2-related phosphosites, Cdc37 pSer13 and NFκB pSer529, was demonstrated by Western blot analysis.

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2ß interaction.

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2ß in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α(1-335) and the fluorescent probe CF-Ahx-Pc as a CK2ß analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α(1-335). Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2ß. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α(1-335) complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α(1-335) were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2ß, with an IC50 value of 92µM.

Identification of a novel potent, selective and cell permeable inhibitor of protein kinase CK2 from the NIH/NCI Diversity Set Library.

The anti-apoptotic protein kinase CK2 increasingly becomes an attractive target in cancer research with great therapeutic potential. Here, we have performed an in vitro screening of the Diversity Set III of the DTP program from the NCI/NIH, comprising 1600 compounds. We have identified 1,3-Dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl] dibenzo(b,d) furan-2,7-diol (referred to as D11) to be a potent and selective inhibitor of protein kinase CK2. The D11 compound was tested against 354 eukaryotic protein kinases. By setting the threshold for inhibition to <2% remaining kinase activity, only DYRK1B, IRAK1 and PIM3 were inhibited to an extent as the tetrameric CK2 holoenzyme and its catalytic subunits α and α'. The IC50 values for the CK2α and CK2α' were on average 1-2 nM in comparison to the DYRK1B, IRAK1 and PIM3 kinases, which ranged from 18 to 49 nM. Cell permeability and efficacy of D11 were tested with cells in culture. In MIA PaCa-2 cells (human pancreatic carcinoma cell line), the phosphorylation of the CK2 biomarker CDC37 at S13 was almost completely inhibited in the presence of D11. This was observed both under normoxia and hypoxia. In the case of the human non-small cell lung carcinoma cell line, H1299, increasing amounts of D11 led to an inhibition of S380/T382/383 phosphorylation in PTEN, another biomarker for CK2 activity.

ATP sensitive bi-quinoline activator of the AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) regulates cellular and whole-body energy balance in response to changes in adenylate charge and hormonal signals. Activation of AMPK in tissues such as skeletal muscle and liver reverses many of the metabolic defects associated with obesity and Type 2 diabetes. Here we report a bi-quinoline (JJO-1) that allosterically activates all AMPK αßγ isoforms in vitro except complexes containing the γ3 subunit. JJO-1 does not directly activate the autoinhibited α subunit kinase domain and differs among other known direct activators of AMPK in that allosteric activation occurs only at low ATP concentrations, and is not influenced by either mutation of the γ subunit adenylate-nucleotide binding sites or deletion of the ß subunit carbohydrate-binding module. Our findings indicate that AMPK has multiple modes of allosteric activation that may be exploited to design isoform-specific activators as potential therapeutics for metabolic diseases.

Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET(2).

Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET(2) pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP(2). This BRET(2) sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET(2) sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate ß-arrestin2, decreased the BRET(2) signal by up to 50%. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50%. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET(2) sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.

Comparative analysis of stress responses of H9c2 rat cardiomyoblasts following treatment with doxorubicin and tBOOH.

Cardiotoxicity is the major dose-limiting adverse effect of anthracyclines and is hypothesized to result from damage induced by reactive oxygen species (ROS) or inhibition of topoisomerase II. Here, we comparatively analyzed the effect of doxorubicin and the organic peroxide tertiary-butylhydroperoxide (tBOOH) on stress responses of rat cardiomyblast cells (H9c2). Moreover, we investigated the impact of serum factors and the novel prototypical protein kinase CK2 inhibitor resorufin on the sensentivity of H9c2 cells exposed to doxorubicin or tBOOH. Measuring cell viability by use of the WST assay as well as cell cycle progression and apoptotic death by FACS-based methods, we found that the sensitivity of H9c2 cells to doxorubicin and tBOOH was differently affected by both serum factors and resorufin. Formation of reactive oxygen species was observed after exposure of H9c2 cells to high doses (i.e. ≥5 µM) of doxorubicin only. Moreover, the antioxidant N-acetylcysteine protected H9c2 cells from cytotoxicity provoked by tBOOH but not doxorubicin. Analyzing the phosphorylation level of genotoxic stress responsive protein kinases and histone H2AX, which is indicative of an activated DNA damage response (DDR), we found that resorufin modulates doxorubicin- and tBOOH-induced responses in an agent specific manner. Taken together, the data indicate that (i) oxidative injury is not the most relevant type of damage triggering cell death of H9c2 cells following doxorubicin treatment, (ii) serum factors differently influence the sensitivity of cardiomyoblasts to doxorubicin and tBOOH and (iii) inhibition of CK2 unequally affects doxorubicin- and tBOOH-induced DDR of rat cardiomyoblasts.

Low-density crystal packing of human protein kinase CK2 catalytic subunit in complex with resorufin or other ligands: a tool to study the unique hinge-region plasticity of the enzyme without packing bias.

A low-resolution structure of the catalytic subunit CK2α of human protein kinase CK2 (formerly known as casein kinase 2) in complex with the ATP-competitive inhibitor resorufin is presented. The structure supplements previous human CK2α structures in which the interdomain hinge/helix αD region adopts a closed conformation correlating to a canonically established catalytic spine as is typical for eukaryotic protein kinases. In the corresponding crystal packing the hinge/helix αD region is nearly unaffected by crystal contacts, so that largely unbiased conformational adaptions are possible. This is documented by published human CK2α structures with the same crystal packing but with an open hinge/helix αD region, one of which has been redetermined here with a higher symmetry. An overview of all published human CK2α crystal packings serves as the basis for a discussion of the factors that determine whether the open or the closed hinge/helix αD conformation is adopted. Lyotropic salts in crystallization support the closed conformation, in which the Phe121 side chain complements the hydrophobic catalytic spine ensemble. Consequently, genuine ligand effects on the hinge/helix αD conformation can be best studied under moderate salt conditions. Ligands that stabilize either the open or the closed conformation by hydrogen bonds are known, but a general rule is not yet apparent.

A subnanomolar fluorescent probe for protein kinase CK2 interaction studies.

Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α.

Interaction between CK2α and CK2ß, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface.

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2ß). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2ß as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2ß. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2ß-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2ß interactions is important for the future generation of small molecule drug design.

Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design.

At the first glance CK2alpha, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2alpha the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2alpha structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2alpha structures. In the latter a surprising plasticity of important ATP-binding elements - the interdomain hinge region and the glycine-rich loop - was discovered. In fully active CK2alpha the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the gamma-phospho group from its functional position. In addition to this partially inactive state human CK2alpha was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2beta: if CK2beta blocks this site, the fully active conformation of CK2alpha is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2α and its paralogous isoform CK2α'.

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α'). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open ß4/ß5-loop. Comparisons of these structures with existing CK2α/CK2α' and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α' an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α'-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2ß. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.

Role of polyamines in determining the cellular response to chemotherapeutic agents: modulation of protein kinase CK2 expression and activity.

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

Lack of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is accompanied by increased CK2α' levels.

DNA-PKcs is the catalytic subunit of DNA-dependent protein kinase, an enzyme necessary for non-homologous end-joining (NHEJ) and hence repair of DNA double strand breaks. Characterization of two isogenic cell lines, M059K and M059J, which are DNA-PKcs-proficient and -deficient, respectively, revealed that lack of DNA-PKcs is accompanied by an increase in the protein level of one of the catalytic isozymes of protein kinase CK2, i.e., CK2α' and a concomitant increase in CK2 activity. The increase was also detectable at the mRNA level as measured by quantitative real time PCR. However, no increase at the DNA level was observed either by comparative PCR or fluorescent in situ hybridization indicating that gene amplification is not involved. Interestingly, only CK2α' was increased and not the other two subunits of CK2, i.e., CK2ß or CK2α. In addition, the increase in CK2α' protein level was also observed in a DNA-PKcs-deficient mouse cell line.

Phytochemicals in cancer and their effect on the PI3K/AKT-mediated cellular signalling.

Protein kinases belong to the largest family of enzymes controlling every aspect of cellular activity including gene expression, cell division, differentiation and metabolism. They are part of major intracellular signalling pathways. Hence, it is not surprising that they are involved in the development of major diseases such as cardiovascular disorders, diabetes, dementia and, most importantly, cancer when they undergo mutations, modifications and unbalanced expression. This review will explore the possibility to draw a connection between the application of natural phytochemicals and the treatment of cancer. We have chosen to focus on the PI3K/AKT cellular signalling pathway which has been shown to be a major target by natural compounds in cell cultures and animal models.

Role of Protein Kinase CK2 in Aberrant Lipid Metabolism in Cancer.

Uncontrolled proliferation is a feature defining cancer and it is linked to the ability of cancer cells to effectively adapt their metabolic needs in response to a harsh tumor environment. Metabolic reprogramming is considered a hallmark of cancer and includes increased glucose uptake and processing, and increased glutamine utilization, but also the deregulation of lipid and cholesterol-associated signal transduction, as highlighted in recent years. In the first part of the review, we will i) provide an overview of the major types of lipids found in eukaryotic cells and their importance as mediators of intracellular signaling pathways ii) analyze the main metabolic changes occurring in cancer development and the role of oncogenic signaling in supporting aberrant lipid metabolism and iii) discuss combination strategies as powerful new approaches to cancer treatment. The second part of the review will address the emerging role of CK2, a conserved serine/threonine protein kinase, in lipid homeostasis with an emphasis regarding its function in lipogenesis and adipogenesis. Evidence will be provided that CK2 regulates these processes at multiple levels. This suggests that its pharmacological inhibition combined with dietary restrictions and/or inhibitors of metabolic targets could represent an effective way to undermine the dependency of cancer cells on lipids to interfere with tumor progression.

The CK2 alpha/CK2 beta interface of human protein kinase CK2 harbors a binding pocket for small molecules.

The Ser/Thr kinase CK2 (previously called casein kinase 2) is composed of two catalytic chains (CK2 alpha) attached to a dimer of noncatalytic subunits (CK2 beta). CK2 is involved in suppression of apoptosis, cell survival, and tumorigenesis. To investigate these activities and possibly affect them, selective CK2 inhibitors are required. An often-used CK2 inhibitor is 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In a complex structure with human CK2 alpha, DRB binds to the canonical ATP cleft, but additionally it occupies an allosteric site that can be alternatively filled by glycerol. Inhibition kinetic studies corroborate the dual binding mode of the inhibitor. Structural comparisons reveal a surprising conformational plasticity of human CK2 alpha around both DRB binding sites. After local rearrangement, the allosteric site serves as a CK2 beta interface. This opens the potential to construct molecules interfering with the CK2 alpha/CK2 beta interaction.

Resorufin: a lead for a new protein kinase CK2 inhibitor.

Screening a natural compound library led to the identification of resorufin as a highly selective and potent inhibitor of protein kinase CK2. Out of 52 kinases tested, only CK2 was inhibited, in contrast to emodin, a structurally related, known CK2 inhibitor that, in addition to CK2, inhibited ten other kinases by 90%. The IC50 values determined for the CK2 holoenzymes were 1.5 mol/l and for the free catalytic subunits ca. 4 mol/l. Altogether four cell lines were subjected to resorufin and emodin treatment. In the case of the three prostate carcinoma cell lines (PC-3, DU-145, LNCaP), 24 h treatment with 40 mol/l resorufin led to 15-20% dead cells; however, no caspase-mediated apoptosis was observed. In the case of the colorectal carcinoma HCT116 cell line, a similar picture was obtained, yet, when resorufin was administered to cells treated with doxorubicin, apoptosis was strongly induced within 24 h. Endogenous protein kinase CK2 was inhibited by resorufin by ca. 80% in the three prostate cell lines. In the case of the HCT116 cells, the inhibition was only 40% supporting the notion of cell line-specific selectivity. Moreover, we analysed the effect of resorufin and emodin on selected signalling molecules in the cell lines under investigation.

Natural Compounds and Derivatives as Ser/Thr Protein Kinase Modulators and Inhibitors.

The need for new drugs is compelling, irrespective of the disease. Focusing on medical problems in the Western countries, heart disease and cancer are at the moment predominant illnesses. Owing to the fact that ~90% of all 21,000 cellular proteins in humans are regulated by phosphorylation/dephosphorylation it is not surprising that the enzymes catalysing these reactions (i.e., protein kinases and phosphatases, respectively) have attracted considerable attention in the recent past. Protein kinases are major team players in cell signalling. In tumours, these enzymes are found to be mutated disturbing the proper function of signalling pathways and leading to uncontrolled cellular growth and sustained malignant behaviour. Hence, the search for small-molecule inhibitors targeting the altered protein kinase molecules in tumour cells has become a major research focus in the academia and pharmaceutical companies.

Delivery of proteins encapsulated in chitosan-tripolyphosphate nanoparticles to human skin melanoma cells.

We have successfully encapsulated two proteins, bovine serum albumin (BSA) and p53, in chitosan-tripolyphosphate (TPP) nanoparticles at various pH values from 5.5 to 6.5 and delivered the particles to human melanoma cells. The particles have diameters ranging from 180 nm to 280 nm and a zeta potential of +15 to + 40 mV. Cellular uptake of the particles by human skin melanoma cells was evaluated by: (i) fluorescence microscopy and (ii) gel electrophoresis showing that FITC-labeled BSA and p53 could be recovered in the soluble cell fraction after lysis of the cells. Our data also show that the highest cellular uptake takes place at the lowest pH as the particles have the highest positive charge under these conditions. The method we describe appears to be a general method for delivery of proteins to cells using chitosan-TPP nanoparticles as a drug delivery system, since structurally unrelated proteins such as BSA and p53 with different isoelectrical points can be encapsulated in the chitosan-TPP nanoparticles and be effectively internalized by the cells.

Identification of multiple post-translational modifications in the porcine brain specific p25alpha.

P25alpha is a protein normally expressed in oligodendrocytes and subcellular relocalization of p25alpha occurs in multiple system atrophy, Parkinson's disease and Lewy body dementia along with ectopic expression in neurons. Moreover, it accumulates in Lewy body inclusions with aggregated alpha-synuclein and is a potent stimulator of alpha-synuclein aggregation. P25alpha is a phosphoprotein and post-translational modifications (PTMs) may play a role in its disease-related abnormalities. To investigate the spectrum of PTMs on p25alpha we cloned porcine p25alpha and isolated the protein from porcine brain. Using several complementary tandem mass spectrometry techniques for peptide mass analysis and amino acid sequencing, a comprehensive analysis of the PTMs on porcine p25alpha was performed. It was found that porcine p25alpha is heavily modified with a variety of modifications: phosphorylation, di- and trimethylation, citrullination and a HexNAc group. The modifications are localized within p25alpha's unfolded terminal domains and suggest that their functional states are regulated. This comprehensive mapping of p25alpha's PTMs will form the basis for future functional studies and investigations of p25alpha's potential role as a biomarker.