JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2006
Sherri-Ann M Burnett MD
Abstract FGF-23 is a novel regulator of phosphate metabolism. We studied the regulation of FGF-23 by dietary phosphate in 66 men and women using two assays. Dietary phosphate restriction decreased FGF-23 and loading increased FGF-23 significantly. An assay that measured intact FGF-23 showed the effects of dietary phosphate much more clearly than an assay that also measures presumed biologically inactive fragments. Dietary phosphate is a key regulator of circulating FGF-23; choice of assay is critical when studying FGF-23 physiology. Introduction: Fibroblast growth factor 23 (FGF-23) is a novel phosphaturic factor discovered through genetic studies of patients with renal phosphate wasting disorders. Ablation of the FGF-23 gene in mice reduces renal phosphate excretion and increases serum phosphate, suggesting that FGF-23 is critical for normal phosphate homeostasis. We examined the role of dietary phosphate in the regulation of FGF-23 in humans. Materials and Methods: Sixty-six healthy males and females were randomized to either phosphate-depleted or -loaded diets for 5 days, after a 4-day run-in diet. FGF-23 was measured using an "intact" assay that only detects intact FGF-23 peptide and with a "C-terminal" assay that measures both intact FGF-23 peptide and presumed biologically inactive carboxyl terminal fragments. The main outcome was the within group change in FGF-23 with either phosphate depletion or loading. Results: Using the intact FGF-23 assay, mean FGF-23 area under the curve (AUC) decreased by 9 ± 16% with phosphate depletion (p = 0.0041) and increased by 35 ± 29% with loading (p < 0.0001). Using the C-terminal FGF-23 assay, mean FGF-23 AUC decreased by 8 ± 12% with phosphate depletion (p = 0.0003) and increased by 13 ± 20% with loading (p = 0.0016). Increases in FGF-23 with phosphate loading were greater with the intact assay than with the C-terminal assay (p = 0.0003). Using the intact assay only, FGF-23 was significantly associated with serum phosphate (r = 0.39, p < 0.01), 24-h urinary phosphate (r = 0.47, p < 0.01), fractional excretion of phosphate (r = 0.29, p < 0.01), and 1,25-dihydroxyvitamin D (r = ,0.30, p < 0.01). The association between the assays was weak (r = 0.26, p < 0.01). Conclusions: Dietary phosphate is a key regulator of circulating FGF-23 levels in humans. Additionally, choice of assay is critical when performing physiologic investigations of FGF-23. [source]

Osteoclast Inhibitory Peptide 2 Inhibits Osteoclast Formation via Its C-Terminal Fragment

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2001
Sun Jin Choi
Abstract Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged complementary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, and the "mature enzyme" form of OIP-2. The proform or the CTF portion of OIP-2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP-2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP-2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF-,B (RANK) ligand. Binding studies with His-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP-2, rather than the mature enzyme, mediates the inhibitory effects of OIP-2 through a putative receptor on OCL precursors. [source]

Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain

CYTOSKELETON, Issue 4 2004
Scott C. Mateer
Abstract IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, ,-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5,50 ,M Kd) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP12-210, which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP12-210 was found to be monomeric, to bind F-actin with a Kd of ,47 ,M, to saturate F-actin at a molar ratio of one IQGAP12-210 per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. Cell Motil. Cytoskeleton 58:231,241, 2004. © 2004 Wiley-Liss, Inc. [source]

Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanism

DEVELOPMENTAL DYNAMICS, Issue 4 2004
Gary R. Hime
Abstract Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function. Developmental Dynamics 229:780,790, 2004. © 2004 Wiley-Liss, Inc. [source]

Voltammetric Studies of the Interactions Between Ferrocene-Labeled Glutathione and Proteins in Solution or Immobilized onto Surface

ELECTROANALYSIS, Issue 16 2009
Yong Peng
Abstract Glutathione (GSH) tagged with a ferrocene (Fc) label at its C-terminal was synthesized via coupling ferrocenyl amine to glutathione using o -(benzotriazol-1-yl)- N,N,N,,N, -tetramethyluronium (HBTU)/1-hydroxybenzotrizole (HOBt). The presence of Fc yielded well defined voltammetric signals, rendering the Fc-tagged GSH (GSH-Fc) suitable for electrochemical studies of GSH binding to other biological species. The interaction of GSH-Fc with bovine serum albumin (BSA) was investigated, and a binding ratio of 1.41±0.06 (GSH-Fc/BSA) and an affinity constant Ka of 6.53±2.01×106,M,1 were determined. These results compare well with those measured by fluorescence using untagged GSH, suggesting that the attachment of Fc to GSH does not significantly perturb the GSH structure and binding behavior. By contrasting the binding behavior to several compounds that are known to conjugate to different domains of BSA, the voltammetric study confirmed that GSH-Fc binds at subdomain IIA of BSA with high affinity. The versatility of GSH-Fc for studying GSH binding to surface-confined proteins was also demonstrated with the GSH binding to electroinactive Zn-metallothionein (Zn7 -MT) through hydrogen binding at the region between the Zn7 -MT , and , domains. [source]

The antimicrobial activity of CCL28 is dependent on C-terminal positively-charged amino acids

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2010
Bin Liu
Abstract Several chemokines have been shown to act as antimicrobial proteins, suggesting a direct contribution to innate immune protection. Based on the study of defensins and other antimicrobial peptides, it has been proposed that cationic amino acids in these proteins play a key role in their antimicrobial activity. The primary structure requirements necessary for the antimicrobial activity of chemokines, however, have not yet been elucidated. Using mouse CCL28, we have identified a C-terminal region of highly-charged amino acids (RKDRK) that is essential to the antimicrobial activity of the murine chemokine. Additionally, other positively-charged amino acids in the C-terminus of the protein contribute to the observed antimicrobial effect. Charge reversal and deletion mutations support our hypothesis that C-terminal positively-charged amino acids are essential for the antimicrobial activity of CCL28. Results also demonstrate that although the C-terminal region of the chemokine is essential, it is not sufficient for full antimicrobial activity of CCL28. [source]

Cathepsin X cleaves the ,2 cytoplasmic tail of LFA-1 inducing the intermediate affinity form of LFA-1 and ,-actinin-1 binding

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2009
Zala Jevnikar
Abstract The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the ,2 chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S769, E768 and A767 from the C-terminal of the ,2 cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein ,-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with ,-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration. [source]

The structure and mode of action of different botulinum toxins

EUROPEAN JOURNAL OF NEUROLOGY, Issue 2006
J. O. Dolly
The seven serotypes (A,G) of botulinum neurotoxin (BoNT) are proteins produced by Clostridium botulinum and have multifunctional abilities: (i) they target cholinergic nerve endings via binding to ecto-acceptors (ii) they undergo endocytosis/translocation and (iii) their light chains act intraneuronally to block acetylcholine release. The fundamental process of quantal transmitter release occurs by Ca2+ -regulated exocytosis involving sensitive factor attachment protein-25 (SNAP-25), syntaxin and synaptobrevin. Proteolytic cleavage by BoNT-A of nine amino acids from the C-terminal of SNAP-25 disables its function, causing prolonged muscle weakness. This unique combination of activities underlies the effectiveness of BoNT-A haemagglutinin complex in treating human conditions resulting from hyperactivity at peripheral cholinergic nerve endings. In vivo imaging and immunomicroscopy of murine muscles injected with type A toxin revealed that the extended duration of action results from the longevity of its protease, persistence of the cleaved SNAP-25 and a protracted time course for the remodelling of treated nerve,muscle synapses. In addition, an application in pain management has been indicated by the ability of BoNT to inhibit neuropeptide release from nociceptors, thereby blocking central and peripheral pain sensitization processes. The widespread cellular distribution of SNAP-25 and the diversity of the toxin's neuronal acceptors are being exploited for other therapeutic applications. [source]

Co-expression of C-terminal truncated alpha-synuclein enhances full-length alpha-synuclein-induced pathology

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2010
Ayse Ulusoy
Abstract Lewy bodies, which are a pathological hallmark of Parkinson's disease, contain insoluble polymers of alpha-synuclein (,syn). Among the different modifications that can promote the formation of toxic ,syn species, C-terminal truncation is among the most abundant alterations in patients with Parkinson's disease. In vitro, C-terminal truncated ,syn aggregates faster and sub-stoichiometric amounts of C-terminal truncated ,syn promote aggregation of the full-length ,syn (,synFL) and induce neuronal toxicity. To address in vivo the putative stimulation of ,syn-induced pathology by the presence of truncated ,syn, we used recombinant adeno-associated virus to express either ,synFL or a C-terminal truncated ,syn (1-110) in rats. We adjusted the recombinant adeno-associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of ,syn were co-expressed at these pre-determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C-terminal truncated ,syn (1-110) but only ,synFL, we demonstrated that the co-expressed truncated protein promoted the progressive accumulation of ,synFL and formation of larger pathological accumulations. Moreover, in the co-expression group, three of the eight animals showed apomorphine-induced turning, suggesting prominent post-synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C-terminal truncated ,syn can interact with and exacerbate the formation of pathological accumulations containing ,synFL in vivo. [source]

Val-Ala Dipeptide Isosteres by Hydrocyanation of ,,-Amino ,,,-Unsaturated Ketones , Control of Stereoselectivity by the N -Protecting Group

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 10 2003
Fabio Benedetti
Abstract Three diastereoisomeric hydroxyethylene isosters of the Val-Ala dipeptide were synthesized from ,,,-unsaturated ketones 1 derived from N -Boc- and N,N -dibenzyl- L -valine. The enones were hydrocyanated with diethylaluminum cyanide to give the corresponding ,-cyano ketones with the stereoselectivity depending on the protecting group. N -Boc protected enone 1a gave a 1:1 mixture of anti and syn adducts 4a, 5a while the corresponding N,N -dibenzyl compound 1c gave a 6:1 mixture of anti, syn adducts 4c, 5c. Borohydride reduction of the resulting cyano ketones is also controlled by the protecting group, resulting in opposite stereoselectivities for N -Boc and N,N -dibenzyl compounds. The cyano alcohols thus obtained were converted, in several steps, into two series of enantiomerically pure hydroxyethylene isosters of the Val-Ala dipeptide. In the first series the hydroxy group and the N -terminal of the isoster are internally protected through the formation of an oxazolidine; in the second series the hydroxy group and the C-terminal are protected as lactone. Two oxazolidines (28, 29), corresponding to syn,syn and syn,anti 4-hydroxy-5-amino acid isosters, and three lactones (23,25), corresponding to syn,syn, syn,anti, and anti,anti isosters were obtained by this approach. (© Wiley-VCH Verlag GmbH & Co KGaA, 69451 Weinheim, Germany, 2003) [source]

Proteolytic activation and function of the cytokine Spätzle in the innate immune response of a lepidopteran insect, Manduca sexta

FEBS JOURNAL, Issue 1 2010
Chunju An
The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spätzle, which functions in dorsal,ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ because of alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the C-terminal 108 residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect the expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and ,-1,3-glucan recognition protein-2. The results of this and other recent studies support a model for a pathway in which the clip-domain proteinase pro-hemolymph proteinase 6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to ,-1,3-glucan. Hemolymph proteinase 6 then activates pro-hemolymph proteinase 8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is likely to function as a ligand to activate a Toll pathway in M. sexta as a response to a wide variety of microbial challenges, stimulating a broad response to infection. Structured digital abstract ,,MINT-7295125: Spätzle 1A (uniprotkb:C8BMD1) and Spätzle 1A (uniprotkb:C8BMD1) bind (MI:0407) by comigration in gel electrophoresis (MI:0807) [source]

Recombinant expression of an insulin-like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3

FEBS JOURNAL, Issue 18 2009
Xiao Luo
Insulin-like peptide 3 (INSL3), which is primarily expressed in the Leydig cells of the testes, is a member of the insulin superfamily of peptide hormones. One of its primary functions is to initiate and mediate descent of the testes of the male fetus via interaction with its G protein-coupled receptor, RXFP2. Study of the peptide has relied upon chemical synthesis of the separate A- and B-chains and subsequent chain recombination. To establish an alternative approach to the preparation of human INSL3, we designed and recombinantly expressed a single-chain INSL3 precursor in Escherichia coli cells. The precursor was solubilized from the inclusion body, purified almost to homogeneity by immobilized metal-ion affinity chromatography and refolded efficiently in vitro. The refolded precursor was subsequently converted to mature human INSL3 by sequential endoproteinase Lys-C and carboxypeptidase B treatment. CD spectroscopic analysis and peptide mapping showed that the refolded INSL3 possessed an insulin-like fold with the expected disulfide linkages. Recombinant human INSL3 demonstrated full activity in stimulating cAMP activity in RXFP2-expressing cells. Interestingly, the activity of the single-chain precursor was comparable with that of the mature two-chain INSL3, suggesting that the receptor-binding region within the mid- to C-terminal of B-chain is maintained in an active conformation in the precursor. This study not only provides an efficient approach for mature INSL3 preparation, but also resulted in the acquisition of a useful single-chain template for additional structural and functional studies of the peptide. [source]

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

FEBS JOURNAL, Issue 8 2008
Camila A. O. Dias
Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal ,-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins , eIF5AK56A and eIF5AQ22H,L93F, and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression. [source]

The Vps4 C-terminal helix is a critical determinant for assembly and ATPase activity and has elements conserved in other members of the meiotic clade of AAA ATPases

FEBS JOURNAL, Issue 7 2008
Parimala R. Vajjhala
Sorting of membrane proteins into intralumenal endosomal vesicles, multivesicular body (MVB) sorting, is critical for receptor down regulation, antigen presentation and enveloped virus budding. Vps4 is an AAA ATPase that functions in MVB sorting. Although AAA ATPases are oligomeric, mechanisms that govern Vps4 oligomerization and activity remain elusive. Vps4 has an N-terminal microtubule interacting and trafficking domain required for endosome recruitment, an AAA domain containing the ATPase catalytic site and a , domain, and a C-terminal , helix positioned close to the catalytic site in the 3D structure. Previous attempts to identify the role of the C-terminal helix have been unsuccessful. Here, we show that the C-terminal helix is important for Vps4 assembly and ATPase activity in vitro and function in vivo, but not endosome recruitment or interactions with Vta1 or ESCRT-III. Unlike the , domain, which is also important for Vps4 assembly, the C-terminal helix is not required in vivo for Vps4 homotypic interaction or dominant-negative effects of Vps4,E233Q, carrying a mutation in the ATP hydrolysis site. Vta1 promotes assembly of hybrid complexes comprising Vps4,E233Q and Vps4 lacking an intact C-terminal helix in vitro. Formation of catalytically active hybrid complexes demonstrates an intersubunit catalytic mechanism for Vps4. One end of the C-terminal helix lies in close proximity to the second region of homology (SRH), which is important for assembly and intersubunit catalysis in AAA ATPases. We propose that Vps4 SRH function requires an intact C-terminal helix. Co-evolution of a distinct Vps4 SRH and C-terminal helix in meiotic clade AAA ATPases supports this possibility. [source]

C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity

FEBS JOURNAL, Issue 23 2007
Chandramouli Reddy Chillakuri
We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and G,i1,1,2 proteins were colocalized in the cells. GTP,S binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the G,i1 protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/G,i1,1,2 complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies. [source]

The C-terminal region of CHD3/ZFH interacts with the CIDD region of the Ets transcription factor ERM and represses transcription of the human presenilin 1 gene

FEBS JOURNAL, Issue 6 2007
Martine Pastorcic
Presenilins are required for the function of ,-secretase: a multiprotein complex implicated in the development of Alzheimer's disease (AD). We analyzed expression of the presenilin 1 (PS1) gene. We show that ERM recognizes avian erythroblastosis virus E26 oncogene homolog (Ets) motifs on the PS1 promoter located at ,10, +90, +129 and +165, and activates PS1 transcription with promoter fragments containing or not the ,10 Ets site. Using yeast two-hybrid selection we identified interactions between the chromatin remodeling factor CHD3/ZFH and the C-terminal 415 amino acids of ERM used as bait. Clones contained the C-terminal region of CHD3 starting from amino acid 1676. This C-terminal fragment (amino acids 1676,2000) repressed transcription of the PS1 gene in transfection assays and PS1 protein expression from the endogenous gene in SH-SY5Y cells. In cells transfected with both CHD3 and ERM, activation of PS1 transcription by ERM was eliminated with increasing levels of CHD3. Progressive N-terminal deletions of CHD3 fragment (amino acids 1676,2000) indicated that sequences crucial for repression of PS1 and interactions with ERM in yeast two-hybrid assays are located between amino acids 1862 and 1877. This was correlated by the effect of progressive C-terminal deletions of CHD3, which indicated that sequences required for repression of PS1 lie between amino acids 1955 and 1877. Similarly, deletion to amino acid 1889 eliminated binding in yeast two-hybrid assays. Testing various shorter fragments of ERM as bait indicated that the region essential for binding CHD3/ZFH is within the amino acid region 96,349, which contains the central inhibitory DNA-binding domain (CIDD) of ERM. N-Terminal deletions of ERM showed that residues between amino acids 200 and 343 are required for binding to CHD3 (1676,2000) and C-terminal deletions of ERM indicated that amino acids 279,299 are also required. Furthermore, data from chromatin immunoprecipitation (ChIP) indicate that CHD3/ZFH interacts with the PS1 promoter in vivo. [source]

Protein disulfide isomerase family proteins involved in soybean protein biogenesis

FEBS JOURNAL, Issue 3 2007
Hiroyuki Wadahama
Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a,, and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions. [source]

Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase

FEBS JOURNAL, Issue 21 2006
Xun-Cheng Su
DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1,171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1,171 of the E. coli DnaB helicase with significant affinity. [source]

The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membrane

FEBS JOURNAL, Issue 7 2006
Amy J. Baldwin
The protein translocation channel at the outer envelope membrane of chloroplasts (Toc75) is synthesized as a larger precursor with an N-terminal transit peptide. Within the transit peptide of the pea Toc75, a major portion of the 10 amino acid long stretch that contains nine glycine residues was shown to be necessary for directing the protein to the chloroplast outer membrane in vitro[Inoue K & Keegstra K (2003) Plant J34, 661,669]. In order to get insights into the mechanism by which the polyglycine stretch mediates correct targeting, we divided it into three tri-glycine segments and examined the importance of each domain in targeting specificity in vitro. Replacement of the most C-terminal segment with alanine residues resulted in mistargeting the protein to the stroma, while exchange of either of the other two tri-glycine regions had no effect on correct targeting. Furthermore, simultaneous replacement of the N-terminal and middle tri-glycine segments with alanine repeats did not cause mistargeting of the protein as much as those of the N- and C-terminal, or the middle and C-terminal segments. These results indicate that the most C-terminal tri-glycine segment is important for correct targeting. Exchanging this portion with a repeat of leucine or glutamic acid also caused missorting of Toc75 to the stroma. By contrast, its replacement with repeats of asparagine, aspartic acid, serine, and proline did not largely affect correct targeting. These data suggest that relatively compact and nonhydrophobic side chains in this particular region play a crucial role in correct sorting of Toc75. [source]

Conformational properties of bacterial DnaK and yeast mitochondrial Hsp70

FEBS JOURNAL, Issue 12 2005
-helical subdomain, Role of the divergent C-terminal
Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70 shows the highest degree of sequence identity with bacterial DnaK. Although they share a functional mechanism and homologous co-chaperones, they are highly specific and cannot be exchanged between Escherichia coli and yeast mitochondria. To provide a structural basis for this finding, we characterized both proteins, as well as two DnaK/mtHsp70 chimeras constructed by domain swapping, using biochemical and biophysical methods. Here, we show that DnaK and mtHsp70 display different conformational and biochemical properties. Replacing different regions of the DnaK peptide-binding domain with those of mtHsp70 results in chimeric proteins that: (a) are not able to support growth of an E. coli DnaK deletion strain at stress temperatures (e.g. 42 °C); (b) show increased accessibility and decreased thermal stability of the peptide-binding pocket; and (c) have reduced activation by bacterial, but not mitochondrial co-chaperones, as compared with DnaK. Importantly, swapping the C-terminal ,-helical subdomain promotes a conformational change in the chimeras to an mtHsp70-like conformation. Thus, interaction with bacterial co-chaperones correlates well with the conformation that natural and chimeric Hsp70s adopt in solution. Our results support the hypothesis that a specific protein structure might regulate the interaction of Hsp70s with particular components of the cellular machinery, such as Tim44, so that they perform specific functions. [source]

Membrane orientation of laminin binding protein

FEBS JOURNAL, Issue 18 2003
An extracellular matrix bridging molecule of Leishmania donovani
Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m -iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end. [source]

Identification of amino acids in antiplasmin involved in its noncovalent ,lysine-binding-site'-dependent interaction with plasmin

FEBS JOURNAL, Issue 9 2003
Haiyao Wang
The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data. [source]

Relation between domain evolution, specificity, and taxonomy of the ,-amylase family members containing a C-terminal starch-binding domain

FEBS JOURNAL, Issue 4 2003
tefan Jane
The ,-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): ,-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic ,-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (,/,)8 -barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the ,complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the ,SBD tree' the border between hydrolases (mainly,-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic ,-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (,/,)8 -barrel(domain A). [source]

A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone

FEBS JOURNAL, Issue 1 2003
Shin-ichi Yamada
The , isoform of human 90-kDa heat shock protein (HSP90,) is composed of three domains: the N-terminal (residues 1,400); middle (residues 401,615) and C-terminal (residues 621,732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650,697 of HSP90, were essential for the binding to the middle domain. An almost identical region (residues 657,720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662,678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90, and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins. [source]

Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coli

FEBS JOURNAL, Issue 21 2002
Sushil Prasad Sati
The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal ,solubilization tag'. This system may allow large-scale production of those proteins that have a tendency to misfold during expression. [source]

Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts

FEBS JOURNAL, Issue 3 2001
Ruth M. Mould
Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of 35S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system. [source]

Intracellular site of ,-secretase cleavage for A,42 generation in Neuro 2a cells harbouring a presenilin 1 mutation

FEBS JOURNAL, Issue 7 2000
Shinji Sudoh
Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid ,-protein (A,)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing A,42,,-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of A,42,,-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP,C100 encoding the C-terminal ,-amyloid precursor protein (APP) fragment truncated at the N terminus of A, (C100); C100 requires only ,-secretase cleavage to yield A,. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced A,1,42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of A,1,40 was not increased. The intracellular generation of A,1,42 from transiently expressed C100 in both mutated PS1 -induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of A,1,42 and A,1,40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of ,-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of A,1,42/40 from C100 was not influenced by monensin treatment, and the level of A,1,42/40 generated from C100 carrying a sorting signal for the trans -Golgi network was higher than that generated from wild-type C100. These results using PS1 -mutation-harbouring and wild-type Neuro 2a cells suggest that A,42/40,,-secretase cleavages occur in the Golgi compartment and the trans -Golgi network, and that the PS1 mutation does not alter the intracelluar site of A,42,,-secretase cleavage in the normal APP proteolytic processing pathway. [source]

Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cells

FEBS JOURNAL, Issue 3 2000
Stavit Drori
Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3,-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3,-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3,-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating , 65,90 kDa native reduced folate carrier and a , 40,45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed. [source]

The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance

FEMS MICROBIOLOGY REVIEWS, Issue 2-3 2003
Laura S. Busenlehner
Abstract The SmtB/ArsR family of prokaryotic metalloregulatory transcriptional repressors represses the expression of operons linked to stress-inducing concentrations of di- and multivalent heavy metal ions. Derepression results from direct binding of metal ions by these homodimeric ,metal sensor' proteins. An evolutionary analysis, coupled with comparative structural and spectroscopic studies of six SmtB/ArsR family members, suggests a unifying ,theme and variations' model, in which individual members have evolved distinct metal selectivity profiles by alteration of one or both of two structurally distinct metal coordination sites. These two metal sites are designated ,3N (or ,3) and ,5 (or ,5C), named for the location of the metal binding ligands within the known or predicted secondary structure of individual family members. The ,3N/,3 sensors, represented by Staphylococcus aureus pI258 CadC, Listeria monocytogenes CadC and Escherichia coli ArsR, form cysteine thiolate-rich coordination complexes (S3 or S4) with thiophilic heavy metal pollutants including Cd(II), Pb(II), Bi(III) and As(III) via inter-subunit coordination by ligands derived from the ,3 helix and the N-terminal ,arm' (CadCs) or from the ,3 helix only (ArsRs). The ,5/,5C sensors Synechococcus SmtB, Synechocystis ZiaR, S. aureus CzrA, and Mycobacterium tuberculosis NmtR form metal complexes with biologically required metal ions Zn(II), Co(II) and Ni(II) characterized by four or more coordination bonds to a mixture of histidine and carboxylate ligands derived from the C-terminal ,5 helices on opposite subunits. Direct binding of metal ions to either the ,3N or ,5 sites leads to strong, negative allosteric regulation of repressor operator/promoter binding affinity, consistent with a simple model for derepression. We hypothesize that distinct allosteric pathways for metal sensing have co-evolved with metal specificities of distinct ,3N and ,5 coordination complexes. [source]

The transcarboxylase domain of pyruvate carboxylase is essential for assembly of the peroxisomal flavoenzyme alcohol oxidase

FEMS YEAST RESEARCH, Issue 7 2007
Paulina Z. Ozimek
Abstract Pyruvate carboxylase (Pyc1p) has multiple functions in methylotrophic yeast species. Besides its function as an enzyme, Pyc1p is required for assembly of peroxisomal alcohol oxidase (AO). Hence, Pyc1p-deficient cells share aspartate auxotrophy (Asp,) with a defect in growth on methanol as sole carbon source (Mut,). To identify regions in Hansenula polymorpha Pyc1p that are required for the function of HpPyc1p in AO assembly, a series of random mutations was generated in the HpPYC1 gene by transposon mutagenesis. Upon introduction of 18 mutant genes into the H. polymorpha PYC1 deletion strain (pyc1), four different phenotypes were obtained, namely Asp, Mut,, Asp, Mut+, Asp+ Mut,, and Asp+ Mut+. One mutant showed an Asp+ Mut, phenotype. This mutant produced HpPyc1p containing a pentapeptide insertion in the region that links the conserved N-terminal biotin carboxylation domain (BC) with the central transcarboxylation (TC) domain. Three mutants that were Asp, Mut, contained insertions in the TC domain, suggesting that this domain is important for both functions of Pyc1p. Analysis of a series of constructed C-terminal and N-terminal truncated versions of HpPyc1p showed that the TC domain of Pyc1p, including the region linking this domain to the BC domain, is essential for AO assembly. [source]