Calcium carbonate polymorph selection in fish otoliths: A key role of phosphorylation of Starmaker-like protein.

Fish otoliths are calcium carbonate biominerals found in the inner ear commonly used for tracking fish biochronologies and as a model system for biomineralization. The process of fish otolith formation is biologically controlled by numerous biomacromolecules which not only affect crystal size, shape, mechanical properties, but also selection of calcium carbonate polymorph (e.g., aragonite, vaterite). The proteinaceous control over calcium carbonate polymorph selection occurs in many other species (e.g., corals, mollusks, echinoderms) but the exact mechanism of protein interactions with calcium and carbonate ions - constituents of CaCO3 - are not fully elucidated. Herein, we focus on a native Starmaker-like protein isolated from vaterite asteriscus otoliths from Cyprinus carpio. The proteomic studies show the presence of the phosphorylated protein in vaterite otoliths. In a series of in vitro mineralization experiments with Starmaker-like, we show that native phosphorylation is a crucial determinant for the selection of a crystal's polymorphic form. This is the first report showing that the switch in calcium carbonate phase depends on the phosphorylation pattern of a single isolated protein. STATEMENT OF SIGNIFICANCE: Calcium carbonate has numerous applications in industry and medicine. However, we still do not understand the mechanism of biologically driven polymorph selection which results in specific biomineral properties. Previous work on calcium carbonate biominerals showed that either several macromolecular factors or high magnesium concentration (non-physiological) are required for proper polymorph selection (e.g., in mollusk shells, corals and otoliths). In this work, we showed for the first time that protein phosphorylation is a crucial factor for controlling the calcium carbonate crystal phase. This is important because a single protein from the otolith organic matrix could switch between polymorphs depending on the phosphorylation level. It seems that protein post-translational modifications (native, not artificial) are more important for biomolecular control of crystal growth than previously considered.

Interplay between hippocampal TACR3 and systemic testosterone in regulating anxiety-associated synaptic plasticity.

Tachykinin receptor 3 (TACR3) is a member of the tachykinin receptor family and falls within the rhodopsin subfamily. As a G protein-coupled receptor, it responds to neurokinin B (NKB), its high-affinity ligand. Dysfunctional TACR3 has been associated with pubertal failure and anxiety, yet the mechanisms underlying this remain unclear. Hence, we have investigated the relationship between TACR3 expression, anxiety, sex hormones, and synaptic plasticity in a rat model, which indicated that severe anxiety is linked to dampened TACR3 expression in the ventral hippocampus. TACR3 expression in female rats fluctuates during the estrous cycle, reflecting sensitivity to sex hormones. Indeed, in males, sexual development is associated with a substantial increase in hippocampal TACR3 expression, coinciding with elevated serum testosterone and a significant reduction in anxiety. TACR3 is predominantly expressed in the cell membrane, including the presynaptic compartment, and its modulation significantly influences synaptic activity. Inhibition of TACR3 activity provokes hyperactivation of CaMKII and enhanced AMPA receptor phosphorylation, associated with an increase in spine density. Using a multielectrode array, stronger cross-correlation of firing was evident among neurons following TACR3 inhibition, indicating enhanced connectivity. Deficient TACR3 activity in rats led to lower serum testosterone levels, as well as increased spine density and impaired long-term potentiation (LTP) in the dentate gyrus. Remarkably, aberrant expression of functional TACR3 in spines results in spine shrinkage and pruning, while expression of defective TACR3 increases spine density, size, and the magnitude of cross-correlation. The firing pattern in response to LTP induction was inadequate in neurons expressing defective TACR3, which could be rectified by treatment with testosterone. In conclusion, our study provides valuable insights into the intricate interplay between TACR3, sex hormones, anxiety, and synaptic plasticity. These findings highlight potential targets for therapeutic interventions to alleviate anxiety in individuals with TACR3 dysfunction and the implications of TACR3 in anxiety-related neural changes provide an avenue for future research in the field.

SIRT1 and HSP90α feed-forward circuit safeguards chromosome segregation integrity in diffuse large B cell lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets exhibit phenotypic features independent of the genetic background. For example, a subset of DLBCLs is distinguished by increased oxidative phosphorylation and unique transcriptional features, including overexpression of certain mitochondrial genes and a molecular chaperone, heat shock protein HSP90α (termed "OxPhos" DLBCLs). In this study, we identified a feed-forward pathogenetic circuit linking HSP90α and SIRT1 in OxPhos DLBCLs. The expression of the inducible HSP90α isoform remains under SIRT1-mediated regulation. SIRT1 knockdown or chemical inhibition reduced HSP90α expression in a mechanism involving HSF1 transcription factor, whereas HSP90 inhibition reduced SIRT1 protein stability, indicating that HSP90 chaperones SIRT1. SIRT1-HSP90α interaction in DLBCL cells was confirmed by co-immunoprecipitation and proximity ligation assay (PLA). The number of SIRT1-HSP90α complexes in PLA was significantly higher in OxPhos- dependent than -independent cells. Importantly, SIRT1-HSP90α interactions in OxPhos DLBCLs markedly increased in mitosis, suggesting a specific role of the complex during this cell cycle phase. RNAi-mediated and chemical inhibition of SIRT1 and/or HSP90 significantly increased the number of cells with chromosome segregation errors (multipolar spindle formation, anaphase bridges and lagging chromosomes). Finally, chemical SIRT1 inhibitors induced dose-dependent cytotoxicity in OxPhos-dependent DLBCL cell lines and synergized with the HSP90 inhibitor. Taken together, our findings define a new OxPhos-DLBCL-specific pathogenetic loop involving SIRT1 and HSP90α that regulates chromosome dynamics during mitosis and may be exploited therapeutically.

Intermitochondrial cement (IMC) harbors piRNA biogenesis machinery and exonuclease domain-containing proteins EXD1 and EXD2 in mouse spermatocytes.

BACKGROUND: Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. AIMS: In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. RESULTS: The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. CONCLUSION: Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

Lipid anchors are common post-translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C-termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP-Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the structural basis of plant REP-Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule-specific, and in other cases they are common for a subset of Rabs. In particular, the C-terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C-terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.

Polyaminoacids in Biomimetic Collagen Mineralization: Roles of Isomerization and Disorder in Polyaspartic and Polyglutamic Acids.

The extracellular matrix of hard connective tissues is composed primarily of mineralized collagen fibrils. Acidic noncollagenous proteins play important roles in mediating mineralization of collagen. Polyaspartate, a homopolymer substitute for such proteins, has been used extensively in in vitro models to produce biomimetic mineralized collagen. Polyglutamate behaves differently in mineralization models, despite its chemical similarity. We show that polyaspartate is a 350 times more effective inhibitor of solution precipitation of hydroxyapatite than polyglutamate. Supersaturated CaP solutions stabilized with polyaspartic acid produce collagen with aligned intrafibrillar mineral, while solutions containing polyglutamate lead to the formation of unaligned mineral clusters on the fibril surface. Molecular analysis showed that the commercial polyaspartic acid contains substantial isomerization, unlike polyglutamic acid. Hence, the secondary structure of polyaspartic acid is more disordered than that of polyglutamic acid. The increased flexibility of the polyaspartic acid chain may explain its potency as an inhibitor of solution crystallization and a mediator of intrafibrillar collagen mineralization.

FORTIS: a live-cell assay to monitor AMPA receptors using pH-sensitive fluorescence tags.

The real-time live fluorescent monitoring of surface AMPA receptors (AMPARs) could open new opportunities for drug discovery and phenotypic screening concerning neuropsychiatric disorders. We have developed FORTIS, a tool based on pH sensitivity capable of detecting subtle changes in surface AMPARs at a neuronal population level. The expression of SEP-GluA1 or pHuji-GluA1 recombinant AMPAR subunits in mammalian neurons cultured in 96-well plates enables surface AMPARs to be monitored with a microplate reader. Thus, FORTIS can register rapid changes in surface AMPARs induced by drugs or genetic modifications without having to rely on conventional electrophysiology or imaging. By combining FORTIS with pharmacological manipulations, basal surface AMPARs, and plasticity-like changes can be monitored. We expect that employing FORTIS to screen for changes in surface AMPARs will accelerate both neuroscience research and drug discovery.

IDH2 mutations in patients with normal karyotype AML predict favorable responses to daunorubicin, cytarabine and cladribine regimen.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients' outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37-0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.

Glycosaminoglycans accelerate biomimetic collagen mineralization in a tissue-based in vitro model.

Mammalian teeth are attached to the jawbone through an exquisitely controlled mineralization process: unmineralized collagen fibers of the periodontal ligament anchor directly into the outer layer of adjoining mineralized tissues (cementum and bone). The sharp interface between mineralized and nonmineralized collagenous tissues makes this an excellent model to study the mechanisms by which extracellular matrix macromolecules control collagen mineralization. While acidic phosphoproteins, localized in the mineralized tissues, play key roles in control of mineralization, the role of glycosaminoglycans (GAGs) is less clear. As several proteoglycans are found only in the periodontal ligament, it has been hypothesized that these inhibit mineralization of collagen in this tissue. Here we used an in vitro model based on remineralization of mouse dental tissues to determine the role of matrix GAGs in control of mineralization. GAGs were selectively removed from demineralized mouse periodontal sections via enzymatic digestion. Proteomic analysis confirmed that enzymatic GAG removal does not significantly alter protein content. Analysis of remineralized tissue sections by transmission electron microscopy (TEM) shows that GAG removal reduced the rate of remineralization in mineralized tissues compared to the untreated control, while the ligament remained unmineralized. Protein removal with trypsin also reduced the rate of mineralization, but to a lesser extent than GAG removal, despite a much larger effect on protein content. These results indicate that GAGs promote mineralization in mineralized dental tissues rather than inhibiting mineral formation in the ligament, which may have broader implications for understanding control of collagen mineralization in connective tissues.

In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Otoliths are one of the biominerals whose formation is highly controlled by proteins. The first protein discovered to be involved in otolith biomineralization in zebrafish was starmaker (Stm). Previously, Stm was shown to be responsible for the preferential formation of aragonite, a polymorph of calcium carbonate, in otoliths. In this work, proteomic analysis of adult zebrafish otoliths was performed. Stm is the only highly phosphorylated protein found in our studies. Besides previously studied otolith proteins, we discovered several dozens of unknown proteins that reveal the likely mechanism of biomineralization. A comparison of aragonite and vaterite otoliths showed similarities in protein composition. We observed the presence of Stm in both types of otoliths. In vitro studies of 2 characteristic Stm fragments indicated that the DS-rich region has a special biomineralization activity, especially after phosphorylation.-Kalka, M., Markiewicz, N., Ptak, M., Sone, E. D., Ozyhar, A., Dobryszycki, P., Wojtas, M. In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Expression of vitamin D receptor in the porcine uterus and effect of 1,25(OH)2D3 on progesterone and estradiol-17ß secretion by uterine tissues in vitro.

Vitamin D3 acting via its nuclear receptor (VDR) was shown to target many reproductive tissues and regulate their function. Nevertheless, little is known about the role of vitamin D3 and VDR in the uterus. We hypothesized that VDR expression profile varies in the porcine uterus throughout the course of the estrous cycle, and 1,25(OH)2D3 influences uterine steroidogenic activity. The aim of this study was to investigate VDR mRNA expression, VDR protein abundance and immunolocalization in the porcine endometrium and myometrium harvested on Days 2-5, 12-13, 15-16 and 18-20 of the estrous cycle. Additionally, in studied pigs, 25OHD concentration in plasma and uterine flushings was determined by RIA. The effect of 1,25(OH)2D3 (10, 50 and 100 ng/mL) in vitro on progesterone (P4) and estradiol-17ß (E2) release by endometrial and myometrial slices obtained on Days 12-13 of the estrous cycle was also examined. Nuclear VDR immunostaining was found in endometrial (luminal and glandular epithelium, stromal cells) and myometrial cells throughout examined days of the estrous cycle. In the endometrium, the highest VDR mRNA expression was observed on Days 12-13 and 18-20, whereas the greatest VDR protein abundance was noted only on Days 12-13 of the estrous cycle. In the myometrium, either VDR transcript or protein level was the greatest on Days 12-13. Interestingly, the highest 25OHD concentration in plasma and uterine flushings was shown also on Days 12-13 of the estrous cycle. 1,25(OH)2D3 did not affect P4 release by uterine slices while myometrial release of E2 was significantly increased in response to 1,25(OH)2D3 (10 and 50 ng/mL). Overall, obtained results indicate that porcine uterus is a target tissue for vitamin D3 throughout the entire estrous cycle. VDR mRNA expression and protein abundance altered within uterine tissues depending on studied days of the estrous cycle with the greatest protein abundance during mid-luteal phase of the estrous cycle in both uterine tissues. In addition, 1,25(OH)2D3 significantly increased myometrial release of E2 on Days 12-13 of the estrous cycle. These results suggest the role of vitamin D3-VDR system in the uterus, especially as a regulator of myometrial estrogenic activity in pigs during mid-luteal phase of the estrous cycle.

pH-Induced transformation of ligated Au25 to brighter Au23 nanoclusters.

Thiolate-protected gold nanoclusters have recently attracted considerable attention due to their size-dependent luminescence characterized by a long lifetime and large Stokes shift. However, the optimization of nanocluster properties such as the luminescence quantum yield is still a challenge. We report here the transformation of Au25Capt18 (Capt labels captopril) nanoclusters occurring at low pH and yielding a product with a much increased luminescence quantum yield which we have identified as Au23Capt17. We applied a simple method of treatment with HCl to accomplish this transformation and we characterized the absorption and emission of the newly created ligated nanoclusters as well as their morphology. Based on DFT calculations we show which Au nanocluster size transformations can lead to highly luminescent species such as Au23Capt17.

Effect of calcium ions on structure and stability of the C1q-like domain of otolin-1 from human and zebrafish.

Otolin-1 is a collagen-like protein expressed in the inner ear of vertebrates. It provides an organic scaffold for otoliths in fish and otoconia in land vertebrates. In this study, the expression and purification procedure of C1q-like domain of otolin-1 from human and zebrafish was developed. The structure and stability of the proteins were investigated. The results of sedimentation velocity analytical ultracentrifugation and small-angle X-ray scattering indicated that the C1q-like domain of otolin-1 forms stable trimers in solution in the presence of calcium ions. It was also observed that calcium ions influenced the secondary structure of the proteins. C1q-like domains were stabilized by the calcium ions. The human variant was especially affected by the calcium ions. The results indicate the importance of the C1q-like domain for the assembly of the organic matrix of otoliths and otoconia.

Regulation of m6A Transcripts by the 3'→5' RNA Helicase YTHDC2 Is Essential for a Successful Meiotic Program in the Mammalian Germline.

N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. High-throughput mapping of the m6A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m6A-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 3'→5' RNA helicase activity. Furthermore, YTHDC2 recruits the 5'→3' exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.

Single-molecule electrometry.

Mass and electrical charge are fundamental properties of biological macromolecules. Although molecular mass has long been determined with atomic precision, a direct and precise determination of molecular charge remains an outstanding challenge. Here we report high-precision (<1e) measurements of the electrical charge of molecules such as nucleic acids, and globular and disordered proteins in solution. The measurement is based on parallel external field-free trapping of single macromolecules, permits the estimation of a dielectric coefficient of the molecular interior and can be performed in real time. Further, we demonstrate the direct detection of single amino acid substitution and chemical modifications in proteins. As the electrical charge of a macromolecule strongly depends on its three-dimensional conformation, this kind of high-precision electrometry offers an approach to probe the structure, fluctuations and interactions of a single molecule in solution.

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 µM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 µM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Intrinsically disordered and pliable Starmaker-like protein from medaka (Oryzias latipes) controls the formation of calcium carbonate crystals.

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

Calponin-like Chd64 is partly disordered.

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function.

Lysine methylation mapping of crenarchaeal DNA-directed RNA polymerases by collision-induced and electron-transfer dissociation mass spectrometry.

Enzymatic machineries fundamental for information processing (e.g., transcription, replication, translation) in Archaea are simplified versions of their eukaryotic counterparts. This is clearly noticeable in the conservation of sequence and structure of corresponding enzymes (see for example the archaeal DNA-directed RNA polymerase (RNAP)). In Eukarya, post-translational modifications (PTMs) often serve as functional regulatory factors for various enzymes and complexes. Among the various PTMs, methylation and acetylation have been recently attracting most attention. Nevertheless, little is known about such PTMs in Archaea, and cross-methodological studies are scarce. We examined methylation and N-terminal acetylation of endogenously purified crenarchaeal RNA polymerase from Sulfolobus shibatae (Ssh) and Sulfolobus acidocaldarius (Sac). In-gel and in-solution protein digestion methods were combined with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) mass spectrometry analysis. Overall, 20 and 26 methyl-lysines for S. shibatae and S. acidocaldarius were identified, respectively. Furthermore, two N-terminal acetylation sites for each of these organisms were assessed. As a result, we generated a high-confidence data set for the mapping of methylation and acetylation sites in both Sulfolobus species, allowing comparisons with the data previously obtained for RNAP from Sulfolobus solfataricus (Sso). We confirmed that all observed methyl-lysines are on the surface of the RNAP.