The DNA-binding domain of the Chd1 chromatin-remodelling enzyme contains SANT and SLIDE domains

The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes. The presence of these domains in ISWI and Chd1 chromatin-remodelling enzymes may provide a means of efficiently harnessing the action of the Snf2-related ATPase domain for the purpose of nucleosome spacing and provide an explanation for partial redundancy between these proteins. Site directed mutagenesis was used to identify residues important for DNA binding and generate a model describing the interaction of this domain with DNA. Through inclusion of Chd1 sequences in homology searches SLIDE domains were identified in CHD6-9 proteins. Point mutations to conserved amino acids within the human CHD7 SLIDE domain have been identified in patients with CHARGE syndrome.     

Adsorption of viral matrix protein M1 in acidic medium

Adsorption of viral matrix protein M1 on the self-assembled monolayer of carboxyhexadecanthiol molecules simulating the surface of the cell membrane was studied by surface plasmon resonance refractometry technique. It was shown that in the acidic medium (pH 4.0) the fraction of irreversibly adsorbed protein increases with time. The protein formed a monolayer on the surface in concentration range from 50 to 500 nM. It was found that the amount of the adsorbed protein increased more than 3 times in this range. An important observation is that even at the lowest concentrations of the protein its molecules totally occupied the entire surface of the substrate, and a further protein addition did not lead to its further adsorption. To explain this phenomena, it was suggested that the number of M1 bonds with the surface increases during the adsorption, which leads to spreading of the protein molecules. Apparently, this effect is caused by the intrinsic disorder of the C-domain of the protein. It is hypothesized that the disassembly of the protein-lipid envelope of the influenza virus in the acidic medium does not result from desorption of the M1, but it is caused by the weakening of protein-protein bonds.     

The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.     

Effects of acidic and basic macromolecules on the activity of protein phosphatase-1

The dephosphorylation of phosphorylase a by the catalytic subunit of protein phosphatase-1 obtained from rabbit skeletal muscle is inhibited by heparin in a noncompetitive manner with respect to phosphorylase a (Ki = 8 micrograms/ml). The inhibitory effect of heparin is also observed in the presence of effectors (e.g., glucose and AMP) modifying the dephosphorylation of phosphorylase a. Heat-stable protein inhibitors of protein phosphatase-1 can develop their inhibitory effect of the activity of protein phosphatase-1 even in the presence of heparin. The inhibitory effect of heparin and the heat-stable inhibitor-2 of phosphatase is additive. Polybrene, a heparin antagonist, prevented phosphatase-1 from the inhibition caused by heparin or the inhibitors. Proteins with basic character, histone fractions (H1, H3) and protamine sulfate, can counteract with the inhibitory effect of heparin, but they cannot intercept the actions of inhibitor-1 or -2.     

New allelic product of the PRH1 locus coding for salivary acidic proline-rich proteins

A new polymorphic acidic proline-rich protein (As) was found in human parotid saliva by SDS and basic polyacrylamide gel electrophoresis. The phenotypic relationships and family studies support the hypothesis that the As protein is another allelic product of the PRH1 locus. The As protein could not be discriminated from the parotid isoelectric focusing (PIf) protein by isoelectric focusing gel electrophoresis due to similar migration of the two proteins. In order to determine salivary PRH1 phenotypes it is necessary to use SDS or basic gel electrophoresis in addition to the isoelectric focusing gel electrophoresis. The As protein was not found in Caucasians. The allele frequencies of the PRH1 locus in Japanese were PRH1 (double-band protein) = 0.035, PRH1(2) (acidic protein) = 0.193, PRH1(4) (PIf) = 0.751, and PRH1(5) (As) = 0.021.     

大熊猫酸性核糖体磷酸蛋白P1 基因cDNA 克隆及序列分析

运用RT-PCR 技术,从大熊猫的肌肉组织总RNA 中成功克隆了酸性核糖体磷酸蛋白P1 (RPLP1)基因的表达序列,并对其进行了测序及初步分析。结果表明:大熊猫RPLP1 基因的表达序列全长为448 bp,开放阅读框(ORF)为344 bp,编码114 个氨基酸的蛋白质,该蛋白的分子量为11.566 kDa,pI 为4.4,含有3 个酪蛋白激酶Ⅱ磷酸化位点和2 个N - 酰基化位点。进一步分析发现,大熊猫RPLP1 基因的表达序列及其编码的氨基酸序列与已报道的部分哺乳动物具有很高的相似性。      

Mutation of the small acidic tract A1 drastically reduces nucleoplasmin activity

Xenopus laevis nucleoplasmin is a molecular chaperone that mediates sperm decondensation and nucleosome assembly. Nucleoplasmin has three acidic tracts (A1, A2 and A3) and until recent years the long polyglutamic tract A2 was thought to be the binding site for basic proteins. However, the latest publications in this field show that neither A2 nor A3 is indispensable for histone and sperm-specific protein binding. In this work, we show that the mutation of only four acidic amino acid residues of the small A1 tract drastically reduces nucleoplasmin decondensing activity, pointing out this region as the potential binding site for sperm proteins.     

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.     

An acidic protein, YBAP1, mediates the release of YB-1 from mRNA and relieves the translational repression activity of YB-1

Eukaryotic Y-box proteins are nucleic acid-binding proteins implicated in a wide range of gene regulatory mechanisms. They contain the cold shock domain, which is a nucleic acid-binding structure also found in bacterial cold shock proteins. The Y-box protein YB-1 is known to be a core component of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm. Here we disrupted the YB-1 gene in chicken DT40 cells. Through the immunoprecipitation of an epitope-tagged YB-1 protein, which complemented the slow-growth phenotype of YB-1-depleted cells, we isolated YB-1-associated complexes that likely represented general mRNPs in somatic cells. RNase treatment prior to immunoprecipitation led to the identification of a Y-box protein-associated acidic protein (YBAP1). The specific association of YB-1 with YBAP1 resulted in the release of YB-1 from reconstituted YB-1-mRNA complexes, thereby reducing the translational repression caused by YB-1 in the in vitro system. Our data suggest that YBAP1 induces the remodeling of YB-1-mRNA complexes.     

Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.     

The acidic domain of hnRNPQ (NSAP1) has structural similarity to Barstar and binds to Apobec1

Apobec1 edits the ApoB mRNA by deaminating nucleotide C(6666), which results in a codon change from Glutamate to stop, and subsequent expression of a truncated protein. Apobec1 is regulated by ACF (Apobec1 complementation factor) and hnRNPQ, which contains an N-terminal "acidic domain" (AcD) of unknown function, three RNA recognition motifs, and an Arg/Gly-rich region. Here, we modeled the structure of AcD using the bacterial protein Barstar as a template. Furthermore, we demonstrated by in vitro pull-down assays that 6xHis-AcD alone is able to interact with GST-Apobec1. Finally, we performed in silico phosphorylation of AcD and molecular dynamics studies, which indicate conformational changes in the phosphorylated form. The results of the latter studies were confirmed by in vitro phosphorylation of 6xHis-AcD by protein kinase C, mass spectrometry, and spectroscopic analyses. Our data suggest hnRNPQ interactions via its AcD with Apobec1 and that this interaction is regulated by the AcD phosphorylation.     

A Novel PEGylated Liposome-Encapsulated SANT75 Suppresses Tumor Growth through Inhibiting Hedgehog Signaling Pathway

The Hedgehog (Hh) pathway inhibitors have shown great promise in cancer therapeutics. SANT75, a novel compound we previously designed to specially inhibit the Smoothened (SMO) protein in the Hh pathway, has greater inhibitory potency than many of commonly used Hh inhibitors. However, preclinical studies of SANT75 revealed water insolubility and acute toxicity. To overcome these limitations, we developed a liposomal formulation of SANT75 and investigated its antitumor efficacy in vitro and in vivo. We encapsulated SANT75 into PEGylated liposome and the mean particle size distribution and zeta-potential (ZP) of liposomes were optimized. Using the Shh-light2 cell and Gli-GFP or Flk-GFP transgenic reporter zebrafish, we confirmed that liposome-encapsulated SANT75 inhibited Hh activity with similar potency as the original SANT75. SANT75 encapsulated into liposome exerted strong tumor growth-inhibiting effects in vitro and in vivo. In addition, the liposomal SANT75 therapy efficiently improved the survival time of tumor-bearing mice without obvious systemic toxicity. The pathological morphology and immunohistochemistry staining revealed that liposomal SANT75 induced tumor cell apoptosis, inhibited tumor angiogenesis as assessed by CD31 and down-regulated the expression of Hh target protein Gli-1 in tumor tissues. Our findings suggest that liposomal formulated SANT75 has improved solubility and bioavailability and should be further developed as a drug candidate for treating tumors with abnormally high Hh activity.     

Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to "coincidence detection," allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.     

HMGB1 protein inhibits DNA replication in vitro: a role of the acetylation and the acidic tail

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.     

PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic

PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV-1) NEF: We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identified. Mutation of this motif yielded a dominant-negative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.     

The inhibitory effect of HMGB-1 protein on the repair of cisplatin-damaged DNA is accomplished through the acidic domain

The well established inhibitory effect of HMGB-1 on repair of cisplatin-damaged DNA has been studied with two modified forms of the protein, shown to bind platinated DNA with higher affinity than the original protein: in vivo acetylated HMGB-1 and HMGB-1 lacking its C-terminal domain. The native and the modified proteins were assayed for their effects on adduct removal by using cell-free extract capable of repairing cisplatinated DNA in vitro. The inhibition observed with the native HMGB-1 was reduced in the presence of acetylated HMGB-1 and completely abolished when the assay was carried out with the truncated protein. When the repair assay was performed in the presence of a synthetic polypeptide identical to the C-terminal tail, either alone or together with the truncated protein, the inhibitory effect was partially recovered in a concentration-dependent manner. These findings strongly suggest that the HMGB-1-induced inhibition of cisplatin-DNA adduct repair is accomplished through the acidic domain. The results obtained are discussed in terms of the repair events that may occur in the presence of HMGB-1 protein.