Chromatin domain boundaries： insulators and beyond

The eukaryotic genome is organized into functionally and structurally distinct domains, representing regulatory units for gene expression and chromosome behavior. DNA sequences that mark the border between adjacent domains are the insulators or boundary elements, which are required in maintenance of the function of different domains. Some insulators need others enable to play insulation activity. Chromatin domains are defined by distinct sets of post-translationally modified histones. Recent studies show that these histone modifications are also involved in establishment of sharp chromatin boundaries in order to prevent the spreading of distinct domains. Additionally, in some loci, the high-order chromatin structures for long-range looping interactions also have boundary activities, suggesting a correlation between insulators and chromatin loop domains. In this review, we will discuss recent progress in the field of chromatin domain boundaries.     

Dissociation of the α-subunit Calf-2 domain and the β-subunit I-EGF4 domain in integrin activation and signaling

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αβ transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αβ legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling.     

The PASTA domain: a β-lactam-binding domain

The PASTA domain (for penicillin-binding protein and serine/threonine kinase associated domain) is found in the high molecular weight penicillin-binding proteins and eukaryotic-like serine/threonine kinases of a range of pathogens. We describe this previously uncharacterized domain and infer that it binds β-lactam antibiotics and their peptidoglycan analogues. We postulate that PknB-like kinases are key regulators of cell-wall biosynthesis. The essential function of these enzymes suggests an additional pathway for the action of β-lactam antibiotics.     

1H, 13C and 15N assignments of Sgt2 N-terminal dimerisation domain and its binding partner, Get5 Ubiquitin-like domain

The first stage of the GET (guided entry of tail-anchored proteins) mechanism for tail-anchored (TA) membrane protein insertion is thought to occur when Sgt2 (small, glutamine-rich, tetratricopeptide repeat-containing protein 2) binds TA proteins upon their release from the ribosome. It sorts them and passes the majority over to a complex of Get5 and Get4 for transmission along the GET pathway and delivery to their membrane destination. Sgt2 is a 38 kDa protein consisting of three domains. The N-terminal domain effects tight dimerisation of the protein and is also the site for binding with the ubiquitin-like (UBL) domain of Get5. Here we have expressed and purified uniformly-¹⁵N/¹³C-labelled N-terminal Sgt2 (Sgt2_NT) and its binding partner, Get5 UBL domain (Get5_UBL) and assigned the backbone and side-chain resonances as a basis for structure solution of the individual components and, ultimately, the complex. This will provide detailed molecular insight into the early stages of the GET pathway.     

Protein folding and three-dimensional domain swapping: a strained relationship?

Many proteins function as multimeric assemblies into which the folded individual promoters organize as higher order structures. An oligomerization mechanism that appears to impose the coordination of events during folding and oligomer assembly is three-dimensional domain swapping. Recent studies have focused on revealing the structural basis of domain swapping and a possible role for domain swapping in the regulation of protein aggregation and activity.     

Reuse of structural domain–domain interactions in protein networks

Background  

Protein interactions are thought to be largely mediated by interactions between structural domains. Databases such as iPfam relate interactions in protein structures to known domain families. Here, we investigate how the domain interactions from the iPfam database are distributed in protein interactions taken from the HPRD, MPact, BioGRID, DIP and IntAct databases.     

Comparison of the homologous carboxy-terminal domain and tail of α-crystallin and small heat shock protein

The C-terminal domain and tail, which is the most conserved region of the -crystallin/small heat shock protein (HSP) family, was obtained from rat A-crystallin, bovine B-crystallin and mouse HSP25. All three domains have primarily -sheet conformation and less than 10% of -helix, like the proteins from which they are derived. Whereas the C-terminal part of A-crystallin forms dimers or tetramers, the corresponding regions of B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the -crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.Abbreviations A2Dt residues 64–173 of rat -crystallin - B2Dt residues 70–175 of bovine B-crystallin - bp base pair - HSP2Dt residues 92–209 of HSP25 - HSP(s) heat shock protein(s) - HSP25 mouse small HSP - PCR polymerase chain reaction - PMSF phenylmethylsulfonyl chloride - SDS sodium dodecyl sulfate; polyacrylamide - WSF water-soluble fraction     

Reversible transition between α-helix and β-sheet conformation of a transmembrane domain

Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP/synaptobrevin and syntaxin 1 are implicated in membrane fusion. Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold into an α-helix inserted at ∼ 35° to the normal of the membranes, an observation in line with molecular simulations. Upon an increase in the peptide/lipid ratio, the peptides readily exhibited transition to β-sheet structure. Moreover, the insertion angle of these β-sheets increased to 54° and was accompanied by a derangement of lipid acyl chains. For both proteins the transition from α-helix to β-sheet was reversible under certain conditions by increasing the peptide/lipid ratio. This phenomenon was observed in different model systems including multibilayers and small unilamellar vesicles. In addition, differences in peptide structure and transitions were observed when using distinct lipids (DMPC, DPPC or DOPC) thus indicating parameters influencing transmembrane domain structure and conversion from helices to sheets. The putative functional consequences of this unprecedented dynamic behavior of a transmembrane domain are discussed.     

N-cadherin enhances APP dimerization at the extracellular domain and modulates Aβ production

Sequential processing of amyloid precursor protein (APP) by β- and γ-secretase leads to the generation of amyloid-β (Aβ) peptides, which plays a central role in Alzheimer's disease pathogenesis. APP is capable of forming a homodimer through its extracellular domain as well as transmembrane GXXXG motifs. A number of reports have shown that dimerization of APP modulates Aβ production. On the other hand, we have previously reported that N-cadherin-based synaptic contact is tightly linked to Aβ production. In the present report, we investigated the effect of N-cadherin expression on APP dimerization and metabolism. Here, we demonstrate that N-cadherin expression facilitates cis-dimerization of APP. Moreover, N-cadherin expression led to increased production of Aβ as well as soluble APPβ, indicating that β-secretase-mediated cleavage of APP is enhanced. Interestingly, N-cadherin expression affected neither dimerization of C99 nor Aβ production from C99, suggesting that the effect of N-cadherin on APP metabolism is mediated through APP extracellular domain. We confirmed that N-cadherin enhances APP dimerization by a novel luciferase-complementation assay, which could be a platform for drug screening on a high-throughput basis. Taken together, our results suggest that modulation of APP dimerization state could be one of mechanisms, which links synaptic contact and Aβ production.     

RUN and FYVE domain–containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17–positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.     

Solution NMR structure of the helicase associated domain BVU_0683(627–691) from Bacteroides vulgatus provides first structural coverage for protein domain family PF03457 and indicates domain binding to DNA

A high-quality NMR structure of the helicase associated (HA) domain comprising residues 627–691 of the 753-residue protein BVU_0683 from Bacteroides vulgatus exhibits an all α-helical fold. The structure presented here is the first representative for the large protein domain family PF03457 (currently 742 members) of HA domains. Comparison with structurally similar proteins supports the hypothesis that HA domains bind to DNA and that binding specificity varies greatly within the family of HA domains constituting PF03457.     

Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases?

Phosphatidylinositol phosphate kinase (PIPK) catalyzes a key step controlling cellular contents of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂], a critical intracellular messenger involved in vesicle trafficking and modulation of actin cytoskeleton and also a substrate of phospholipase C to produce the two intracellular messengers, diacylglycerol and inositol-1,4,5-trisphosphate. In addition to the conserved C-terminal PIPK catalytic domain, plant PIPKs contain a unique structural feature consisting of a repeat of membrane occupation and recognition nexus (MORN) motifs, called the MORN domain, in the N-terminal half. The MORN domain has previously been proposed to regulate plasma membrane localization and phosphatidic acid (PA)-inducible activation. Recently, the importance of the catalytic domain, but not the MORN domain, in these aspects was demonstrated. These conflicting data raise the question about the function of the MORN domain in plant PIPKs. We therefore performed analyses of PpPIPK1 from the moss Physcomitrella patens to elucidate the importance of the MORN domain in the control of enzymatic activity; however, we found no effect on either enzymatic activity or activation by PA. Taken together with our previous findings of lack of function in plasma membrane localization, there is no positive evidence indicating roles of the MORN domain in enzymatic and functional regulations of PpPIPK1. Therefore, further biochemical and reverse genetic analyses are necessary to understand the biological significance of the MORN domain in plant PIPKs.Key words: membrane occupation and recognition nexus (MORN) domain, phosphatidylinositol phosphate kinase, phosphatidic acid, Physcomitrella patensPhosphoinositides (PIs) are minor membrane phospholipds that play pivotal roles in various signal transduction cascades involved in development and stress response via the regulation of cytoskeletal organization, ion channel activation and vesicle trafficking.^1,2 These are derivatives of phosphatidylinositol (PtdIns) produced by phosphorylation of the 3-, 4- and 5- positions of the inositol ring.² To address the roles of PIs, enzymes involved in their production have been extensively studied using biochemical and molecular biological approaches. Of these enzymes, phosphatidylinositol monophosphate kinases (PIPKs) catalyze the reaction producing phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] that is a substrate of phospholipase C and phosphatidylinositol 3-kinase, and also acts as an intracellular messenger involved in the regulation of F-actin organization and activity of ion channels.^1–3 Although PtdIns(4,5)P₂ is produced by sequential phosphorylation by phosphatidylinositol 4-kinase, producing phosphatidylinositol-4-phosphate [PtdIns(4)P], and then by PIPK,^1,2 the cellular levels of PtdIns(4)P are much higher compared to PtdIns(4,5)P₂.^4–6 Thus, a restriction step controlling cellular PtdIns(4,5)P₂ contents is mediated by PIPKs, indicating the importance of PIPK regulation in various kinds of physiological processes.The roles of plant PIPKs have been established in growth regulation, such as polarized tip growth of root hairs and pollen tubes, via their localization at plasma membranes.^7–12 It is worth to note that plant PIPKs contain a unique structure consisting of a repeat of a membrane occupation recognition nexus (MORN) motifs, called MORN domain, at the N-terminal region and a C-terminal PIPK catalytic domain, except for AtPIP5K10 and AtPIP5K11 from Arabidopsis thaliana, which lack the N-terminal MORN domain.¹³ The MORN domain was first identified as plasma membrane-binding module in junctophilin¹⁴ and the involvement of the MORN domain in plasma membrane localization was proposed for A. thaliana AtPIP5K1 and AtPIP5K3.^9,15,16Another remarkable feature of eukaryotic PIPKs is dependency of the enzymatic activity on phosphatidic acid (PA).^17,18 Indeed, PA-dependent activation of PIPKs has been observed in A. thaliana and in the moss Physcomitrella patens,^6,19,20 as with animal type I PIPKs.²¹ Although much less is known about how PA activates PIPKs in plants, biochemical analyses suggested the involvement of the MORN domain in PA-dependent activation of AtPIP5K1.¹⁵Based on above findings, it was proposed that plasma membrane-localization and PA-dependent activation of plant PIPKs might be regulated by the MORN domain.^9,15,16 In contrast, we recently demonstrated the critical involvement of the C-terminal half containing the catalytic domain of plant PIPKs in both plasma membrane-localization and PA-dependent activation.²² Thus, the function of the MORN domain remains elusive in plant PIPKs.As shown earlier, the N-terminal half of P. patens PpPIPK1 containing the MORN domain enhances its catalytic activity.²² Thus, to identify the region required for the activation of PpPIPK1, we further dissected the N-terminal half into 3 regions; the N-terminal region (amino acid nos. 1–154), the MORN repeat (amino acid nos. 155–316) and the linker region (amino acid nos. 338–452), and made deletion mutants of PpPIPK1 as shown in Figure 1A. Using Pfu Turbo DNA polymerase (Stratagene, La Jolla, USA), DNA fragments corresponding to deletion mutants lacking the N-terminal and N-terminal plus the MORN repeat, designated PpPIPK1ΔN and PpPIPK1ΔN-MORN, respectively, were amplified with primer sets; one is M_PIPK1_fb (5′-GGC AAG CAC GTG TAT AAT GTC TGA AGG GCT T-3′) and XhoIPIPK1 (5′-TAA ACT CGA GTT AGC TGG GTA GGA GGA AA-3′) and the other is M_PIPK1_f7 (5′-AGA GAA CAC GTG TAT AAT GTC TGA CTT CTA CGT CGG T-3′) and XhoIPIPK1. For building an expression plasmid for a deletion mutant lacking the MORN repeat, designated PpPIPK1ΔMORN, the N-terminal region and PpPIPK1ΔN-MORN were amplified with primer sets, M_PIPK1_fb and M_PIPK1_r3 (5′-TTG TAA GTC TCG GGT GCC ATT TGA GAG CTC-3′) M_PIPK1_f6 (5′-GAG CTC TCA AAT GGC ACC CGA GAC TTA CAA-3′) and XhoIPIPK1, respectively, using Pfu Turbo DNA polymerase and resultant DNA fragments were fused by PCR with a primer set, M_PIPK1_fb and XhoIPIPK1 using the same enzyme. These PCR products were digested with Pml1 and XhoI and inserted into Pml1-XhoI digested pPICZB (Invitrogen) to construct expression plasmids, pPICZB-PpPIPK1ΔN, pPICZB-PpPIPK1ΔN-MORN and pPICZB-PpPIPK1ΔMORN. Transformation of P. pastoris X-33 cells with the above expression plasmids, colony PCR of transformants and following expression, purification and western blot analysis of His-tagged recombinant proteins were performed as described previously.⁶ The PIPK activity assay using purified His-tagged proteins was carried out as described previously²³ with the modifications.⁶Open in a separate windowFigure 1Functional dissection of the N-terminal region of PpPIPK1 identifies positive regulatory regions. (A) His-tagged recombinant PpPIPK1 proteins. A repetition of eight MORN motifs (grey boxes) and the conserved catalytic domain (black box) are indicated in wild type and mutant PpPIPK1s. The MORN repeat and junction of internal deletion are indicated by amino acid position numbers. (B) In vitro lipid kinase activity of His-tagged recombinant proteins. The activities of recombinant proteins bound to Ni-NTA agarose beads were assayed with PtdIns4P. (C) In vitro PA-dependent lipid kinase activity of His-tagged proteins. The activities of recombinant proteins bound to Ni-NTA agarose beads were assayed with PtdIns4P with 143 µM PA. Top and bottom arrowheads represent reaction products PtdIns(4,5)P₂ and lysoPtdIns(4,5)P₂, respectively.Biochemical analyses of these enzymes after expression in yeast P. pastoris X-33 cells followed by purification showed that deletion of the N-terminal region (PpPIPK1ΔN) reduced PpPIPK1 activity ca 40% compared to the full length enzyme, whereas loss of the MORN repeat (PpPIPK1ΔMORN) had no significant effect (Fig. 1B). In agreement, a mutant lacking four MORN repeats of the total eight repeats showed no difference in the activity compared the full length enzyme (data not shown). These results indicate a positive role of the N-terminal region, but not the MORN repeats, on PpPIPK1 activity. However, these findings differ from those obtained with AtPIP5K1, where the MORN domain represses enzymatic activity.¹⁵ Interestingly, PpPIPK1ΔN-MORN containing the linker and catalytic regions showed higher enzymatic activity of ca 23 % compared to the full length PpPIPK1 (Fig. 1B). The C-terminal half only containing the catalytic domain of PpPIPK1 and thus lacking the linker region showed a reduced activity.²² It is therefore proposed that the linker region carries a positive regulatory element. Although details are unknown, negligible effects of the N-terminal and MORN domains for the enzymatic activity has been indicated in AtPIP5K3 from A. thaliana.¹¹ Moreover, it is noteworthy that PA-dependent activation was not affected by any deletion as shown in Figure 1C, confirming that the N-terminal half is not involved in PA dependency of the PpPIPK1 activity.²²Our results indicated that the MORN domain is not involved in the regulation of the catalytic activity in PpPIPK1. Similarly, the function of the MORN domain found in the accumulation and replication of chloroplasts 3 (ARC3) was not resolved. ARC3 is an FtsZ homologue involved in chloroplast division²⁴ and the only protein containing the MORN repeats other than PIPKs in A. thaliana. It was shown that the ARC3 MORN domain did not interact with any stromal plastid division components.²⁵ Moreover, there are reports representing functions of the MORN domain other than plasma membrane binding. Human amyotrophic lateral sclerosis 2 (ALS2), a guanine nucleotide exchange factor (GEF) specific to the small GTPase Rab5, contains the MORN domain at the central region that is essential for the GEF activity but not for interaction with Rab5.²⁶ In contrast, specific interaction of the MORN domain with Rab-E GTPases and resultant enzymatic activation was recently demonstrated for AtPIP5K2.¹² It is interesting that these results are inconsistent with each other in terms of interaction of the MORN domain with small GTPases.Taken together, with no function of the MORN domain in plasma membrane localization of PpPIPK1 and AtPIP5K1,²² the function of the MORN domain is still unknown, despite its high conservation plants PIPKs. Alternatively, based on the findings of ARC3, ALS2 and AtPIP5K2,^12,25,26 the function of the MORN domain possibly varies among PIPK isoforms and may thus have multifunctional roles. Therefore, it is necessary to identify interaction partners for the MORN domain of each plant PIPKs and to analyze phenotypes of transgenic plants carrying MORN domain-lacking PIPKs during developmental process and environmental stress responses.     

Fast and reliable prediction of domain–peptide binding affinity using coarse-grained structure models

Domain–peptide recognition and interaction are fundamentally important for eukaryotic signaling and regulatory networks. It is thus essential to quantitatively infer the binding stability and specificity of such interaction based upon large-scale but low-accurate complex structure models which could be readily obtained from sophisticated molecular modeling procedure. In the present study, a new method is described for the fast and reliable prediction of domain–peptide binding affinity with coarse-grained structure models. This method is designed to tolerate strong random noises involved in domain–peptide complex structures and uses statistical modeling approach to eliminate systematic bias associated with a group of investigated samples. As a paradigm, this method was employed to model and predict the binding behavior of various peptides to four evolutionarily unrelated peptide-recognition domains (PRDs), i.e. human amph SH3, human nherf PDZ, yeast syh GYF and yeast bmh 14-3-3, and moreover, we explored the molecular mechanism and biological implication underlying the binding of cognate and noncognate peptide ligands to their domain receptors. It is expected that the newly proposed method could be further used to perform genome-wide inference of domain–peptide binding at three-dimensional structure level.     

cDNA cloning and expression analysis of a novel human F-box domain containing gene†

F-box proteins are a large family of eukaryotic proteins that are characterized by an approximately 40 amino acid motif. Some F-box proteins are critical for the controlled degradation of cellular regulatory proteins. Here we report that a novel member of F-box proteins, FBXO35 gene, was cloned and identified during the large-scale sequencing analysis from a human fetal brain cDNA library. FBXO35 gene shares amino acid similarity with several putative mouse genes not only in F-box domain but also in the rest of the sequence, which indicates that FBXO35 might also contain some other unknown conserved domain. RT-PCR analysis indicated that FBXO35 gene had a ubiquitously low expression pattern in most human adult tissues. According to bioinformatics analysis, the FBXO35 gene was found located in chromosome 3p21.     

Both the N-terminal fragment and the protein–protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP

Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.     

Direct and indirect determination of tibial natural frequency — A comparison of frequency domain analysis and Fast Fourier Transform

The natural frequency of in vivo mechanical systems has been derived in the past by signal conditioning techniques. For the living tibia the accuracy of this derived method was not validated. In addition the use of Fast Fourier Transform is time consuming, tedious and expensive. Direct measurement yields results which are more accurate, more easily obtained, and more clinically applicable.