Carboxy-terminal Heptapeptide Research Articles

Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.

Fusing the Carboxy-Terminal Heptapeptide of LHβ to FSHβ Induces LH-like Intracellular Behavior of FSH in GH3 Cells.

Fusing the Carboxy-Terminal Heptapeptide of LHβ to FSHβ Induces LH-like Intracellular Behavior of FSH in GH3 Cells.

The carboxy terminus of the small subunit of TFIIE regulates the transition from transcription initiation to elongation by RNA polymerase II.

The general transcription factor TFIIE plays essential roles in both transcription initiation and the transition from initiation to elongation. Previously, we systematically deleted the structural motifs and characteristic sequences of the small subunit of human TFIIE (hTFIIE beta) to map its functional regions. Here we introduced point mutations into two regions located near the carboxy terminus of hTFIIE beta and identified the functionally essential amino acid residues that bind to RNA polymerase II (Pol II), the general transcription factors, and single-stranded DNA. Although most residues identified were essential for transcription initiation, use of an in vitro transcription assay with a linearized template revealed that several residues in the carboxy-terminal helix-loop region are crucially involved in the transition stage. Mutations in these residues also affected the ability of hTFIIE beta to stimulate TFIIH-mediated phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of Pol II. Furthermore, these mutations conspicuously augmented the binding of hTFIIE beta to the p44 subunit of TFIIH. The antibody study indicated that they thus altered the conformation of one side of TFIIH, consisting of p44, XPD, and Cdk-activating kinase subunits, that is essential for the transition stage. This is an important clue for elucidating the molecular mechanisms involved in the transition stage.

A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae.

Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.

Reinforcing effects of neurokinin substance P in the ventral pallidum: mediation by the tachykinin NK 1 receptor

The neurokinin substance P has reinforcing effects when administered into the nucleus basalis of the rat's ventral pallidum and these effects are encoded by its carboxy-terminal amino acid sequence. The present study examined the effect of prior treatment with the tachykinin NK 1 receptor antagonist WIN51,708 on the conditioned place preference produced by intrabasalis injection of substance P and its carboxy-terminal heptapeptide analog dimethyl-C7. Pretreatment with WIN51,708 (10 and 20 mg/kg, i.p.) dose-dependently reversed the place preference produced by intrabasalis substance P (0.74 pmol). The carboxy-terminal analog dimethyl-C7 (0.74 pmol) was also found to act as a reinforcer following injection into the nucleus basalis region, but unlike for substance P, the behavioral effects of dimethyl-C7 could not be completely antagonized by joint administration of the NK 1 antagonist. When injected alone, WIN51,708 did not influence the preference behavior. These findings suggest that the reinforcing effects of substance P in the nucleus basalis region might be mediated via NK 1 receptive sites. The failure of WIN51,708 to completely antagonize the behavioral effects of dimethyl-C7 is interesting in the light of evidence, indicating that the carboxy-terminal substance P analog shows higher affinity for the tachykinin NK 3 than for the NK 1 receptor subtype.

The carboxy-terminal region of the beta-subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimers.

One of the major structural differences between the LH beta and CG beta subunits is the carboxy-terminal region: beyond amino acid 114, LH beta has a hydrophobic heptapeptide stretch, while CG beta contains a 31-amino acid hydrophilic carboxy-terminal peptide (CTP) that is O-glycosylated. The CG beta subunit is secreted quantitatively as a monomer and assembles efficiently whereas secretion and assembly of LH beta is inefficient. We previously implicated the carboxy-terminal heptapeptide as a determinant for the different intracellular behavior manifested by the LH beta subunit compared with the CG beta subunit. Here we tested the function of the heptapeptide and CTP domains by fusing them to their counterparts at amino acid 114 of CG beta or LH beta subunits. The secretion and assembly of these chimeras were examined in transfected Chinese hamster ovary cells. Removal of the heptapeptide enhanced the amount of LH beta subunit secreted 4-fold compared with intact LH beta. Fusion of this heptapeptide to CG beta 114, i.e. CG beta lacking the CTP, decreased the amount of secreted subunit 2-fold compared with wild type human CG beta. Similar experiments reveal that although deleting the CTP from the CG beta subunit did not significantly alter secretion, the combination efficiency of the truncated subunit was reduced to 60%. Perturbing the native carboxy-terminal sequence of either subunit increased the heterogeneity of the secreted forms. This result suggests that these regions are also involved in the posttranslational processing of the asparagine-linked oligosaccharides of the beta-subunits. Fusion of the LH beta heptapeptide to the truncated CG beta subunit decreased combination with the alpha-subunit. These data further support the hypothesis that the carboxy-terminal regions of LH beta and CG beta subunits play a role in the intracellular behavior of the corresponding heterodimers.

Identification of endothelin-1, endothelin-2 and endothelin-3 in human endometrium

This study determined the presence of specific endothelin isoforms in human endometrium using high performance liquid chromatography (HPLC) combined with radioimmunoassay, and immunocytochemistry to detect the endothelin precursors (proendothelin-1, proendothelin-2 and proendothelin-3). Endothelin-like immunoreactivity was measured in HPLC eluates using antisera raised in rabbits against the carboxy-terminal heptapeptide of endothelin-1, which is common to the three endothelin isoforms. Of eight endometrial samples analysed by HPLC, three were in the proliferative phase of the cycle, and five in the secretory phase. Endothelin-1 was detected in seven samples, whereas endothelin-2 and endothelin-3 were seen in four and five specimens, respectively. No relationship was seen between endothelin isoforms and the stage of the cycle. Immunocytochemistry was performed on five proliferative and three secretory phase tissues. When present, staining for the precursor proendothelins was localized to endometrial glandular and luminal epithelium (proendothelin-1, 5 of 8; proendothelin-2, 5 of 8; proendothelin-3, 6 of 8). In two sections, staining was also seen in vascular endothelium using antibody raised against proendothelin-1 (n = 1) and proendothelin-3 (n = 1). These data provide evidence that the three endothelin isoforms are present in human endometrium, and suggest that these potent vasoactive agents may play a role in the paracrine control of the uterine vascular bed.

Endothelin-like immunoreactivity in human endometrium.

Endothelin-like immunoreactivity (ET-IR) was detected immunocytochemically in glandular epithelium and vascular endothelium of human endometrium and myometrium. Primary antibody was raised in rabbits against the carboxy-terminal heptapeptide of endothelin 1 (ET-1), ET-1(15-21), and compared with antibodies raised against the cyclized amino-terminal, ET-1(2-13), and commercially obtained antibodies against the whole ET-1 or ET-3 molecule. Binding was visualized using the peroxidase technique in sections counter-stained with haemalum. Staining was seen in each of 15 sections from eight women in the proliferative (five) or secretory (three) phase of the cycle. Intense staining was present in the cytoplasm of endometrial glands and vascular endothelium, and was greatest at the endometrial-myometrial junction. The pattern of staining was similar with all primary antibodies tested. The demonstration of ET-IR in endometrium suggests that the endothelins may play a role in control of the uterine vascular bed.

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

Active Uptake System for Substance P Carboxy-Terminal Heptapeptide (5-11) into a Fraction from Rabbit Enriched in Glial Cells

In the present study, we demonstrated the existence of an active uptake system for substance P carboxy-terminal heptapeptide, (5–11)SP. When a fraction from rabbit brain enriched in glial cells was incubated with [3H](5–11)SP, an uptake of [3H](5–11)SP was observed. The uptake system has the properties of an active transport mechanism. Kinetic analysis indicated two components of [3H](5–11)SP uptake, one representing a high and the other a low affinity transport system. After unilateral ablation of the striatum, approximately 30% of the high affinity [3H](5–11)SP uptake capacity of substantia nigra slices disappeared. The subcellular distribution of the high affinity uptake indicated that [3H]5-hydroxy-tryptamine was taken up mostly into the P2B fraction (synaptosomal fraction), whereas [3H](5-11)SP was taken up into the P2A fraction (myelin fraction) to the same extent as into the P2B fraction. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5–11)SP, which is in turn accumulated into glial cells as well as nerve terminals and that this high affinity uptake mechanism may play an important role in terminating the synaptic action of SP.

Active uptake system for substance P carboxy-terminal heptapeptide (5-11) into a fraction from rabbit enriched in glial cells.

In the present study, we demonstrated the existence of an active uptake system for substance P carboxy-terminal heptapeptide, (5-11)SP. When a fraction from rabbit brain enriched in glial cells was incubated with [3H] (5-11)SP, an uptake of [3H](5-11)SP was observed. The uptake system has the properties of an active transport mechanism. Kinetic analysis indicated two components of [3H](5-11)SP uptake, one representing a high and the other a low affinity transport system. After unilateral ablation of the striatum, approximately 30% of the high affinity [3H](5-11)SP uptake capacity of substantia nigra slices disappeared. The subcellular distribution of the high affinity uptake indicated that [3H] 5-hydroxytryptamine was taken up mostly into the P2B fraction (synaptosomal fraction), whereas [3H](5-11)SP was taken up into the P2A fraction (myelin fraction) to the same extent as into the P2B fraction. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP, which is in turn accumulated into glial cells as well as nerve terminals and that this high affinity uptake mechanism may play an important role in terminating the synaptic action of SP.

Active uptake of substance P carboxy-terminal heptapeptide (5-11) into rat brain and rabbit spinal cord slices.

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.

The presence of substance P carboxy-terminal heptapeptide in pig brain stem.

The presence of substance P carboxy-terminal heptapeptide in pig brain stem.

Effect of substance P C-terminal heptapeptide on amylase release in rat parotid gland

Substance P carboxy-terminal heptapeptide ([ pGlu 5] SP 5−11) showed a similar potency on the salivation and amylase release in anaesthetized rats to that of substance P. In an in-vitro experiment using parotid slices, the rate of amylase release by [ pGlu 5] SP 5−11 was significantly higher than by substance P. The results suggest that the active heptapeptide may be released from substance P by endogenous peptidase. Baclofen, a substance P antagonist, did not influence the effects of substance P and [ pGlu 5] SP 5−11 in the in-vivo and slice experiments.

Inhibition of Gastric Acid Secretion in Man by Peptide Analogues of Cholecystokinin

In human subjects gastric acid secretion was stimulated by intravenous infusion of 1 μg of pentagastrin per kilogram hour. One hour after the start of pentagastrin, when acid secretion had reached a plateau, either caerulein (eight tests), a decapeptide extracted from frog skins, or carboxy-terminal heptapeptide amide of cholecystokinin (five tests), each in a dose of 0.1 μg per kilogram hour, was added to the pentagastrin infusion for one hour. Both peptides produced about 70 per cent inhibition of acid output. Since both peptides have the same carboxy-terminal tetrapeptide amide sequence as pentagastrin, presumably they inhibited by competing with pentagastrin for a common receptor. In separate tests this dose of caerulein alone produced slight stimulation of acid secretion in two of five subjects. Screening of related peptides might yield a specific competitive inhibitor of gastrin with even less stimulatory action than was noted with those tested.