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PubMed2000

HBV polymerase interacts independently with N-terminal and C-terminal fragments of Hsp90beta.

Hsp90 is an abundant chaperone protein that assists the folding of specific proteins, such as steroid receptors, protein kinases, and so on, for their proper function. TP and RT domains of HBV polymerase have been also shown to be associated with Hsp90. Therefore, the identification of the binding sites within Hsp90, responsible for forming Hsp90/HBV Pol complex, is important for the understanding of HBV replication. In this study, cotransfection and immunoprecipitation experiments were performed to localize the binding sites of HBV pol to Hsp90. Our data show that HBV pol interact independently with both N-terminal and C-terminal fragments of Hsp90. Further analysis showed that N-terminal fragment (1-302) of Hsp90 interacts with both TP and RT domains of HBV pol, whereas C-terminal fragment (438-723) interacts with only RT domain. In conclusion, we showed that HBV pol independently interacts with N-terminal and C-terminal fragments, but not the middle fragment (327-438) of Hsp90.

Animals↗
PubMed2000

Construction of full-length cDNA clones to Soil-borne wheat mosaic virus RNA1 and RNA2, from which infectious RNAs are transcribed In vitro: virion formation and systemic infection without expression of the N-terminal and C-terminal extensions to the capsid protein.

The 19-kDa capsid protein (CP) of Soil-borne wheat mosaic furovirus (SBWMV) is encoded in the 5'-terminal region of RNA2. In addition to CP, two CP-related proteins are translated from SBWMV RNA2: (1) a 24-kDa protein (N-CP) with an N-terminal 40-amino-acid extension initiated at an upstream in-frame CUG codon; and (2) an 83-kDa protein (CP-RT) with an about 580-amino-acid, C-terminal extension by partial translational readthrough at the UGA termination codon at the end of the CP gene. We examined requirements for N-CP and CP-RT on virion formation and systemic infection in wheat plants using full-length cDNA clones, from which infectious RNA can be transcribed in vitro. RNA2 mutants, which could not synthesize N-CP, CP-RT, or either infected wheat plants systemically in combination with the wild-type RNA1 transcripts, produced rod-shaped virus particles in uninoculated upper leaves. Original mutations which abolished translation of N-CP and CP-RT were confirmed on RNA2 extracted from purified virus from the upper leaves by nucleotide sequence analysis. These results indicate that neither N-terminal nor C-terminal extensions to the CP are required for virion formation and systemic infection of SBWMV in wheat plants.

Base Sequence↗
PubMed1982

Chromatin structure of Xenopus rDNA transcription termination sites. Evidence for a two-step process of transcription termination.

The ultrastructure of Xenopus laevis (X.l.) 40S pre-rRNA transcription termination sites was investigated by electron microscopy. Amplified nucleolar chromatin was rapidly dispersed and processed for chromatin spread preparations. This type of preparation revealed that many rDNA termination sites appeared as 50 nm long segments of chromatin axis covered by a complex of three closely spaced RNA polymerase particles. Particle (Ptl) was characterized by the association with the terminal full-length pre-rRNP fibril; particles (Pt2) and (Pt3) are located downstream from (Ptl) and appear to be devoid of transcript fibrils. This particular structural arrangement as well as sequence homology analyses of 3' adjacent spacer rDNA segments indicate that transcript release and dissociation of polymerase particles are not necessarily coupled and termination of X.l. rDNA transcription may occur as a two step process. The structural data are correlated with homologies of DNA sequences at Xenopus rDNA transcription termination regions and are discussed with respect to sequence data of 3' termination sites of rRNA genes from other species.

Animals↗
PubMed1985

Transcription termination within the E1A gene of adenovirus induced by insertion of the mouse beta-major globin terminator element.

In induced erythroleukemia cells, transcription of the beta-globin gene terminates in a region 600-1500 nucleotides downstream of the poly(A) site. To determine whether this region of the mouse DNA functions to terminate transcription when moved to another genomic site, portions of the putative termination region have been inserted into the E1A transcription unit of the adenovirus (type 5) chromosome. Analysis of RNA labeled either in isolated nuclei or in whole cells early after infection with reconstructed viruses indicated that transcription is terminated if the inserted DNA is oriented in the same direction as in the beta-globin transcription unit and contains the globin poly(A) site plus an additional 1395 nucleotides downstream. In addition to halting transcription within the E1A unit, the insertion of the terminator region had a negative cis effect on the E1B transcription unit, which normally initiates 363 bp downstream of the site of the globin insert. The E1B transcription unit was the only early gene affected, and complementation of the terminator virus with a wild-type E1A gene did not restore transcription of the E1B gene.

Adenoviridae↗
PubMed1993

Production of N-terminal and C-terminal human serum transferrin in Escherichia coli.

The elucidation of the relationship of the structure of human serum transferrin to its iron-binding activity and the delineation of the interactions between transferrin and its receptor will require the construction and production of site-specific mutants of human serum transferrin to test the importance of specific structural motifs to the functions of transferrin. The N-terminal domain of transferrin has been previously produced in BHK cells, but the production of the C-terminal domain of transferrin has never been reported. The amino-terminal and carboxyl-terminal half-molecules of human serum transferrin have been cloned into the T7 expression vector pET11a. Contrary to previous reports, nTf and cTf can be easily produced in E. coli. The plasmids produce 38-kDa proteins that are approximately the sizes predicted for N-terminal and C-terminal half-molecules of transferrin, and both proteins react with anti-human serum transferrin antibodies. It is estimated that nTf represents 30-40% of total cellular protein after induction, while cTf represents less than 5% of total cellular protein. This demonstrates that recombinant forms of human serum transferrin can be produced in E. coli and suggests that it will be possible to use a bacterial system to produce other structural variants of transferrin.

Base Sequence↗
PubMedDate not supplied

Substance P and behavior: opposite effects of N-terminal and C-terminal fragments.

Most of the biological actions of substance P (SP) have been thought to be mediated by the carboxy-terminal portion of the peptide. Some of the behavioral effects produced by exogenous SP exhibit a strikingly different structure-activity relationship. The N-terminal heptapeptide fragment of SP, SP(1-7), inhibits nociceptive, aggressive and grooming behaviors and stimulates investigative motor behavior, but the C-terminal hexapeptide fragment analog pyroglutamyl-SP(7-11) exerts opposite effects. While the C-terminal fragment mimics the effects of administered intact SP on motor behaviors, the N-terminal fragment mimics the effects of intact SP on aggressive and nociceptive behaviors. The significant behavioral effects of SP(1-7) and the consistently opposite behavioral effects of N- and C-terminal fragments are important new findings.

Aggression↗
PubMed1982

Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda.

For cloning, assaying the function and sequencing terminators, we have constructed the pD12 plasmid, in which the late promotor p'R of phage lambda controls the expression of the galK gene of the pK03 plasmid of McKenney et al. (1981). The lambda tL3 terminator region was cloned in this plasmid between the promoter and the galK gene, and found to be 90-94% effective in preventing galactokinase expression in both rho+ and rho- hosts. Is is also active in vitro, both in the presence or absence of the rho factor. The termination point is located at 4320 bp to the left of the SL startpoint of the PL-RNA, just downstream of gene exo. We have sequenced 356 bp of the hitherto uncharted lambda DNA to the right of the TaqI cut, which in turn is 110 bp to the right of the b522 deletion at 63.9% lambda. The tL3 terminator has several features common to other rho-independent termination sequences, including an 81% G+C-rich region of 2X8-bp symmetry ("stem") with a 5-bp intervening "loop", partially overlapping and followed by a sequence transcribed into the pyrimidine-rich CCUUUCU-OH 3' terminus of the RNA. The termination point that follows the last U was determined by the S1 mapping technique.

Bacteriophage lambda↗
PubMed1991

Liquid chromatography-mass spectrometry for simultaneous analyses of iminodipeptides containing an N-terminal or a C-terminal proline.

Simultaneous analyses of synthetic iminodipeptides containing an N-terminal proline or a C-terminal proline have been demonstrated using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system. The separation of iminodipeptides was carried out on a reversed-phase high-performance liquid chromatographic column using 0.1% aqueous trifluoroacetic acid-methanol (75:25, v/v, pH 2.0) as mobile phase. Very intense protonated molecular ions [M + H]+ of various synthetic iminodipeptides, Pro-Gly, Gly-Pro, Pro-Ala, Ala-Pro, Pro-Val, Val-Pro, Pro-Leu and Leu-Pro, were observed. Pro-Gly (Pro-X) and Gly-Pro (X-Pro) have the same protonated molecular ion (m/z 173), but the peaks of these compounds on the mass chromatograms were clearly distinguished by the differences of the retention times and mass spectra. The synthetic iminodipeptides containing an N-terminal proline added to urine samples from a patient with prolidase deficiency were also distinguished from iminodipeptides containing a C-terminal proline in urine samples from a patient with prolidase deficiency by scanning the [M + H]+ ion of each iminodipeptide. We established the method to measure simultaneously the various iminodipeptides containing an N-terminal or a C-terminal proline in biological samples.

Amino Acid Sequence↗
PubMed2004

The N-terminal rhodanese domain from Azotobacter vinelandii has a stable and folded structure independently of the C-terminal domain.

Sulfurtransferase are enzymes involved in the formation, conversion and transport of compounds containing sulfane-sulfur atoms. Although the three-dimensional structure of the rhodanese from the nitrogen-fixing bacterium Azotobacter vinelandii is known, the role of its two domains in the protein conformational stability is still obscure. We have evaluated the susceptibility to proteolytic degradation of the two domains of the enzyme. The two domains show different resistance to the endoproteinases and, in particular, the N-terminal domain shows to be more stable to digestion during time than the C-terminal one. Cloning and overexpression of the N-terminal domain of the protein was performed to better understand its functional and structural role. The recombinant N-terminal domain of rhodanese A. vinelandii is soluble in water solution and the spectroscopic studies by circular dichroism and heteronuclear NMR spectroscopy indicate a stable fold of the protein with the expected alpha/beta topology. The results indicate that this N-terminal domain has already got all the elements necessary for an C-terminal domain independent folding. Its solution structure by NMR, actually under course, will be a valid contribution to understand the role of this domain in the folding process of the sulfurtransferase.

Amino Acid Sequence↗
PubMed2005

Adenosine attenuates C-terminal but not N-terminal proteolysis of cTnI during cardioplegic arrest.

BACKGROUND: Specific site proteolysis and loss of troponin I (TnI) during myocardial ischemic events can contribute to myocardial dysfunction. Adenosine supplementation of cardioplegic solutions results in improved functional preservation of the heart. We investigated the effect of adenosine on N-terminal and C-terminal proteolysis of TnI in the heart. MATERIALS AND METHODS: Hearts from male Sprague-Dawley rats were isolated and perfused at a constant pressure. Cardioplegic arrest (St. Thomas #2 +/- 100 microm adenosine) was induced and hearts frozen at various times during the arrest. Antibodies directed against specific regions of TnI were used to visualize TnI in whole heart homogentates, as well as from cellular fractions, using western blot analysis. RESULTS: Cardioplegic arrest alone resulted in early N-terminal proteolysis of TnI, followed by later loss of sequences from the C-terminal end of the molecule. In addition, secondary protein bands that were immunoreactive to amino acid sequences centrally located on the TnI molecule were observed. There was also evidence of dissociation of TnI from the other myofibrillar proteins. The supplementation of cardioplegic solution with adenosine significantly attenuated the late C-terminal proteolytic degradation of TnI and its apparent dissociation from myofibrils proteins but had no effect on the early N-terminal proteolysis associated with cardioplegic arrest. CONCLUSIONS: These data may provide an explanation for partial protection against postarrest myocardial dysfunction provided by adenosine.

Adenosine↗
PubMed2000

Role of the N-terminal region of staphylokinase (SAK): evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complex formation.

Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), which requires both the conversion of PG to plasmin (Pm) to expose an active site in PG-SAK activator complex and the amino-terminal processing of SAK to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of SAK affects its PG activation capability was investigated by generating SAK mutants, blocked in the native amino-terminal processing site of SAK, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilized PG-SAK binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with PG, Pm or miniplasmin substrate during ternary complex formation as compared to native SAK. Additionally, activator complex formed with PRM3 failed to activate miniplasminogen and exhibited highly diminished activation of substrate PG. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of SAK in accession and processing of substrate by the SAK-Pm activator complex to potentiate the PG activation by enhancing and/or stabilizing the interaction of free PG.

Amino Acid Sequence↗
PubMed1999

Substitutions in the C-terminal portion of the catalytic domain partially reverse assembly defects introduced by mutations in the N-terminal linker sequence of cytochrome P450 2C2.

Mutations in a 7-amino acid linker segment, immediately following the N-terminal signal anchor sequence of cytochrome P450 2C2, have been shown to affect proper assembly of hemoprotein and decrease activity of the mutants expressed in COS cells. In contrast, C2pmBalC1, in which cytochrome P450 2C1 residues were substituted for those of cytochrome P450 2C2 in the C-terminal region, exhibited increased activity when expressed in COS-1 cells. To examine further the basis for the increased activity of C2pmBalC1 in COS-1 cells, the protein was expressed in insect cells and Escherichia coli. The amounts of the functional P450 species of C2pmBalC1 expressed in these systems and the ratios of P450 to P420 were greater than those of cytochrome P450 2C2, indicating that more efficient assembly underlies the increased activity of C2pmBalC1. To determine whether the C-terminal substitutions could compensate for the decreased assembly mediated by the N-terminal linker mutations, the linker mutations were introduced into C2pmBalC1. If all 7 amino acids in the linker were deleted, no enzymatically active cytochrome P450 2C2 or C2pmBalC1 was detected in COS-1, insect, or bacterial cells expressing the mutants. The mutant C2A2, in which two alanines were substituted for the linker, had no detectable laurate hydroxylase activity in COS-1 cells, and minor amounts of hemoprotein for this mutant were expressed in E. coli and insect cells. In contrast, the same mutation in C2pmBalC1 reduced activity only 50% in COS-1 cells and markedly elevated levels of P450 expression in bacteria and insect cells. The A2 mutation did not affect the enzymatic activity of either cytochrome P450 2C2 or C2pmBalC1 assayed in whole cell lysates of insect cells but reduced the activity of partially purified enzymes assayed in a reconstituted assay system. These findings indicate that mutations introduced into the C-terminal region of P450 2C2 can facilitate assembly of the proteins and partially reverse the decreased assembly resulting from the N-terminal mutations.

Amino Acid Sequence↗
PubMed1992

Human platelet glycoprotein IIIb binds to thrombospondin fragments bearing the C-terminal region, and/or the type I repeats (CSVTCG motif), but not to the N-terminal heparin-binding region.

Major blood membrane platelet glycoprotein IIIb (GPIIIb), also termed GPIV or CD365, has been identified as a receptor for thrombospondin (TSP), collagen and Plasmodium falciparum-infected erythrocytes. The aim of the present study was to identify region(s) of TSP involved in binding of GPIIIb. Proteolytic fragments of TSP (M(r) 140 kDa, 120-18 kDa and 27 kDa on SDS/PAGE under reducing conditions) were purified by f.p.l.c. and identified by N-terminal gas-phase sequencing, e.l.i.s.a. and Western blots using monoclonal antibodies directed against defined domains of TSP. The 140 kDa and 120-18 kDa fragments (C-terminal region), but not the 27 kDa fragment (N-terminal region), were shown to bind to GPIIIb by using e.l.i.s.a. and affinity-chromatography systems. TSP binding to a GPIIIb-affinity column was Ca(2+)-dependent and reduced by 45% in the presence of EDTA. Moreover, TSP was only partially eluted with EDTA from a Ca(2+)-equilibrated GPIIIb column. A fragment of 68 kDa, obtained by further digestion of the 140 kDa fragment, bound to the GPIIIb-Sepharose affinity column. This fragment, or stalk-like region, bears the TSP type I repeats that show sequence similarity to regions on properdin, Plasmodium falciparum proteins and antistasin. Peptides (CSVTCG or SVTCGGGV) representing these repeats bound isolated GPIIIb in a Ca(2+)-independent way, but did not completely inhibit the GPIIIb and TSP interaction. These studies indicate that GPIIIb binds to the TSP via the C-terminal region and/or the CSVTCG motif, but not to the N-terminal region. Interaction between GPIIIb and the TSP C-terminal region or the CSVTCG motif is respectively Ca(2+)-dependent and -independent.

Amino Acid Sequence↗
PubMed1997

Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity.

It has been suggested that the tethering caused by binding of the N-terminal region of smooth muscle caldesmon (CaD) to myosin and its C-terminal region to actin contributes to the inhibition of actin-filament movement over myosin heads in an in vitro motility assay. However, direct evidence for this assumption has been lacking. In this study, analysis of baculovirus-generated N-terminal and C-terminal deletion mutants of chicken-gizzard CaD revealed that the major myosin-binding site on the CaD molecule resides in a 30-amino acid stretch between residues 24 and 53, based on the very low level of binding of CaDDelta24-53 lacking the residues 24-53 to myosin compared with the level of binding of CaDDelta54-85 missing the adjacent residues 54-85 or of the full-length CaD. As expected, deletion of the region between residues 24 and 53 or between residues 54 and 85 had no effect on either actin-binding or inhibition of actomyosin ATPase activity. Deletion of residues 24-53 nearly abolished the ability of CaD to inhibit actin filament velocity in the in vitro motility experiments, whereas CaDDelta54-85 strongly inhibited actin filament velocity in a manner similar to that of full-length CaD. Moreover, CaD1-597, which lacks the major actin-binding site(s), did not inhibit actin-filament velocity despite the presence of the major myosin-binding site. These data provide direct evidence for the inhibition of actin filament velocity in the in vitro motility assay caused by the tethering of myosin to actin through binding of both the CaD N-terminal region to myosin and the C-terminal region to actin.

Actins↗
PubMed1995

Transcription termination at the thr attenuator. Evidence that the adenine residues upstream of the stem and loop structure are not required for termination.

The Escherichia coli thr operon attenuator has a structure similar to other Rho-independent terminators. The DNA sequence immediately 5' to the termination site is dG+dC-rich and contains a region of dyad symmetry that, when transcribed into RNA, encodes a hairpin structure in the transcript. It also contains a stretch of 9 consecutive dA-dT residues immediately distal to the region of dyad symmetry which encode uridine residues at the 3' end of the terminated transcript. In addition, the thr attenuator has a stretch of 6 dA-dT residues immediately upstream of the region of dyad symmetry which encode 6 adenines. These adenines could potentially pair with the distal uridines to form a hairpin structure extended by as much as 6 A-U base pairs. In this report we have examined the role of the upstream adenines in transcription termination. We used templates that specify mismatches or create new base pairs in the potential A-U secondary structure of the transcript as well as templates that delete segments of the A residues upstream of the hairpin. We conclude that A-U pairing is not required for efficient transcription termination at the thr attenuator. This conclusion is likely to apply to other Rho-independent terminators that contain hairpin-proximal dA-dT residues.

Amino Acid Sequence↗
PubMed1995

Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain.

Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NH2-terminal domain (ezrin 1-233) and that of a COOH-terminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Sf9 cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 microns in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlström, and A. Vaheri. 1994. J. Cell Biol. 126:1445-1453), suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NH2-terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.

Actins↗
PubMed1988

Steric relationship of amino-terminal and carboxy-terminal domains of murine interferon-gamma as assessed by monoclonal antibodies.

Spleen cells from a hamster immunized with murine interferon-gamma (IFN-gamma) carboxy-terminal (95-133) synthetic peptide [IFN-gamma (95-133)] conjugated to keyhole limpet hemocyanin (KLH) were fused with mouse myeloma cells, resulting in the production of an anti-IFN-gamma (95-133) monoclonal antibody, which reacted with IFN-gamma. This monoclonal antibody bound [125I]IFN-gamma in a dose-dependent and reversible fashion, and neutralized the antiviral and macrophage priming activities of IFN-gamma. Antibody-antibody competition for [125I]IFN-gamma binding, using this carboxy-terminal-specific antibody and two previously described amino-terminal-specific monoclonal antibodies, indicated that the carboxy-terminal-specific monoclonal antibody competed only with itself and that the two amino-terminal-specific monoclonal antibodies similarly competed only with each other for [125I]IFN-gamma. The data suggest that the amino- and carboxy-terminal IFN-gamma domains important for function are sterically distant from one another, and suggest the intriguing possibility that interaction of IFN-gamma with its receptor may involve more than one binding site on the IFN-gamma molecule.

Animals↗
PubMed1976

Aging, terminal decline, and terminal drop.

Data from a 20-year longitudinal study of persons over 60 were analyzed by step-wise multiple regression to test for declines in function with age, for terminal decline (linear relationship to time before death), and for terminal drop (curvilinear relationship to time before death). There were no substanial terminal drop effects. All health measures had substantial declines with age, and the physician's physical function rating, as well as the individual's self-rating, showed additional terminal decline. Intelligence had a substantial decline with age and a small terminal decline. Most activities declined with age but had no significant terminal decline. Despite these declines in health, intelligence, and activities, there was little or no decline in satisfaction or adjustment.

Aging↗