John E. Folk

Is hypusine essential for eukaryotic cell proliferation

Hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] occurs in all eukaryotes at one residue in a highly conserved protein, the putative eukaryotic translation initiation factor 5A (eIF-5A, old terminology eIF-4D). This unusual amino acid is produced in a unique posttranslational modification reaction that involves the conjugation of the 4-aminobutyl moiety of the polyamine spermidine to the epsilon-amino group of a specific lysine residue of the eIF-5A precursor protein to form the deoxyhypusine [N epsilon-(4-aminobutyl)lysine] residue and its subsequent hydroxylation. The strict specificity of hypusine synthesis, its derivation from spermidine and its requirement for the activity of eIF-5A and for eukaryotic cell proliferation have raised keen interest in the physiological function of the hypusine-containing protein, eIF-5A.

Nonpeptidic δ-opioid Receptor Agonists Reduce Immobility in the Forced Swim Assay in Rats

The present study examined the effect of opioid receptor agonists in the rat forced swim assay. The δ-opioid receptor agonists SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide) and (+)BW373U86 ((+)-[1(S*),2α,5β]-4-[[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl] (3-hydroxyphenyl)methyl]-N,N-diethyl-benzamide dihydrochloride) produced a decrease in immobility indicating an antidepressant-like effect. At antinociceptive doses, neither the κ-opioid selective agonist CI977 (5R-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl-1-oxaspiro[4,5]dec-8-yl]-4-benzofuranacetamide) showed a change in immobility that was identifiable by dose, nor were changes in immobility seen with morphine. A δ-opioid mechanism of action in the forced swim assay was likely since naltrindole prevented the effects of both δ-agonists. When compared to desipramine and fluoxetine, SNC80 was more active with a single dose whereas both desipramine and fluoxetine produced greater effects with subchronic dosing (3 doses). All three compounds were active when administered before the initial swim exposure. SNC80 was, however, more effective following a single dose than by subchronic administration demonstrating both a fast onset of activity and potential tolerance. Thus, δ-agonists differ from typical antidepressants in the forced swim assay.

Posttranslational formation of hypusine in a single major protein occurs generally in growing cells and is associated with activation of lymphocyte growth

Growing lymphocytes perform a novel chemical modification of a single protein (Hy+: approximately 18 kd, pI approximately 5.1), resulting in the formation of the unusual amino acid, hypusine (N epsilon-[4-amino-2-hydroxybutyl]lysine). This posttranslational event occurs only following activation of lymphocyte growth. Hypusine formation increases at a rate parallel to protein synthesis during the first 24 hr of growth stimulation, beginning before 6 hr of growth. At all times, hypusine is restricted primarily to the single protein, Hy+. In resting cells, the unmodified substrate protein, Hy0, is continuously synthesized and maintained in a steady-state pool of significant size. In several other cell lines, hypusine formation was also observed in a single protein of approximately 18 kd, pI approximately 5.1, indistinguishable electrophoretically from the lymphocyte protein. Thus Hy+ is a ubiquitous protein showing significant conservation among divergent species. Maintenance by resting lymphocytes of a pool of unmodified protein and early activation during growth of the hypusine-forming enzyme system suggest that this posttranslational modification may be of importance to lymphocyte activation.

Nε‐(γ‐l‐Glutamyl)‐l‐lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease

Pathological‐length polyglutamine (Qn) expansions, such as those that occur in the huntingtin protein (htt) in Huntingtons disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post‐mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of Nε‐(γ‐l‐glutamyl)‐l‐lysine (GGEL)– a ‘marker’ isodipeptide produced by the transglutaminase reaction – are elevated in the CSF of HD patients (708 ± 41 pmol/mL, SEM, n = 36) vs. control CSF (228 ± 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

The Convulsive and Electroencephalographic Changes Produced by Nonpeptidic δ-Opioid Agonists in Rats: Comparison with Pentylenetetrazol

δ-Opioid agonists produce convulsions and antidepressant-like effects in rats. It has been suggested that the antidepressant-like effects are produced through a convulsant mechanism of action either through overt convulsions or nonconvulsive seizures. This study evaluated the convulsive and seizurogenic effects of nonpeptidic δ-opioid agonists at doses that previously were reported to produce antidepressant-like effects. In addition, δ-opioid agonist-induced electroencephalographic (EEG) and behavioral changes were compared with those produced by the chemical convulsant pentylenetetrazol (PTZ). For these studies, EEG changes were recorded using a telemetry system before and after injections of the δ-opioid agonists [(+)-4-[(αR)-α-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenz (SNC80) and [(+)-4-[α(R)-α-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide [(+)-BW373U86]. Acute administration of nonpeptidic δ-opioid agonists produced bilateral ictal and paroxysmal spike and/or sharp wave discharges. δ-Opioid agonists produced brief changes in EEG recordings, and tolerance rapidly developed to these effects; however, PTZ produced longer-lasting EEG changes that were exacerbated after repeated administration. Studies with antiepileptic drugs demonstrated that compounds used to treat absence epilepsy blocked the convulsive effects of nonpeptidic δ-opioid agonists. Overall, these data suggest that δ-opioid agonist-induced EEG changes are not required for the antidepressant-like effects of these compounds and that neural circuitry involved in absence epilepsy may be related to δ-opioid agonist-induced convulsions. In terms of therapeutic development, these data suggest that it may be possible to develop δ-opioid agonists devoid of convulsive properties.

The δ -Opioid Receptor Agonist (+)BW373U86 Regulates BDNF mRNA Expression in Rats

δ-Opioid receptor agonists have antidepressant-like effects in behavioral models of depression. Chronic administration of classical antidepressants upregulates mRNA expression of brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor, TrkB in the frontal cortex and hippocampus of rats. Increases in BDNF and TrkB levels are thought to be important for the therapeutic effects of these drugs. Therefore, we examined the ability of the δ-opioid receptor agonist (+)BW373U86 to regulate BDNF and TrkB mRNA expression in frontal cortex, hippocampus, as well as, basolateral amygdala, endopiriform nucleus, and primary olfactory cortex. At 3 h after a single administration of (+)BW373U86 animals were killed and BDNF and TrkB mRNA levels were examined by in situ hybridization. BDNF mRNA levels produced by (+)BW373U86 were compared to acute administration of the antidepressants desipramine and bupropion. A behaviorally antidepressant dose of 10 mg/kg (+)BW373U86 increased BDNF mRNA expression in all regions examined; a smaller dose of (+)BW373U86 (1 mg/kg) significantly increased BDNF mRNA expression only in frontal cortex. The δ-opioid receptor antagonist naltrindole blocked (+)BW373U86-mediated increases in BDNF mRNA expression. In addition, tolerance developed to increased BDNF mRNA expression with repeated injection, except in frontal cortex. Midazolam was administered to some animals to prevent the convulsions produced by (+)BW373U86, but midazolam did not block δ-opioid receptor-mediated increases in BDNF mRNA expression in frontal cortex, hippocampus, or amygdala. Unlike desipramine and bupropion, (+)BW373U86 upregulated BDNF mRNA expression acutely (within 3 h after a single administration). These data support the concept that δ-opioid receptor agonists may have antidepressant potential, and could be good targets for the development of faster-acting antidepressants.

Increased Levels of γ-glutamylamines in Huntington disease CSF

Transglutaminases (TGases) catalyze several reactions with protein substrates, including formation of γ‐glutamyl‐ε‐lysine cross‐links and γ‐glutamylpolyamine residues. The resulting γ‐glutamylamines are excised intact during proteolysis. TGase activity is altered in several diseases, highlighting the importance of in situ enzymatic determinations. Previous work showed that TGase activity (as measured by an in vitro assay) and free γ‐glutamyl‐ε‐lysine levels are elevated in Huntington disease (HD) and that γ‐glutamyl‐ε‐lysine is increased in HD CSF. Although free γ‐glutamyl‐ε‐lysine was used in these studies as an index of in situ TGase activity, γ‐glutamylpolyamines may also be diagnostic. We have devised methods for the simultaneous determination of four γ‐glutamylamines in CSF: γ‐glutamyl‐ε‐lysine, γ‐glutamylspermidine, γ‐glutamylputrescine, and bis‐γ‐glutamylputrescine and showed that all are present in normal human CSF at concentrations of ∼150, 670, 40, and 240 nM, respectively. The high γ‐glutamylspermidine/γ‐glutamylputrescine and γ‐glutamylspermidine/bis‐γ‐glutamylputrescine ratios presumably reflect in part the large spermidine to putrescine mole ratio in human brain. We also showed that all four γ‐glutamylamines are elevated in HD CSF. Our findings support the hypotheses that (i) γ‐glutamylpolyamines are reflective of TGase activity in human brain, (ii) polyamination is an important post‐translational modification of brain proteins, and (iii) TGase‐catalyzed modification of proteins is increased in HD brain.

Covalent incorporation of polyamines as γ-glutamyl derivatives into CHO cell protein

Abstract The possible role of polyamines in the covalent modification of proteins in CHO cells was investigated by metabolic labeling with [ 3 H]putrescine. A single radiolabeled protein band with an apparent relative molecular mass of 18 000 Da was observed by SDS-polyacrylamide gel electrophoresis. Almost all the radioactivity covalently linked to this protein was recovered as hypusine. The labeling of this protein was increased several-fold when cells were cultured with α-difluoromethylornithine (DFMO) or with this drug plus methylglyoxal bis(guanylhydrazone) (MGBG), as a result of increase in specific radioactivity of the hypusine immediate precursor, spermidine. Also labeled under the latter condition were other cellular proteins. These were aggregates on the top both of the stacking gel and of the running gel, and protein-like materials with relative molecular masses of 36 and 8 kDa. The radioactivity covalently associated with these proteins was recovered after acid hydrolofsis as polyamines. The identification of γ-glutamylputrescine and γ-glutamylspermidines in proteolytic digests of the acid-insoluble fraction of treated cells indicates that polyamines are covalently linked to these cellular protein. Several possible cellular functions of γ-glutamylpolyamine protein components are discussed.

The selective non-peptidic delta opioid agonist SNC80 does not facilitate intracranial self-stimulation in rats.

Delta opioid receptor agonists are under development for a variety of clinical applications, and some findings in rats raise the possibility that agents with this mechanism have abuse liability. The present study assessed the effects of the non-peptidic delta opioid agonist SNC80 in an assay of intracranial self-stimulation (ICSS) in rats. ICSS was examined at multiple stimulation frequencies to permit generation of frequency-response rate curves and evaluation of curve shifts produced by experimental manipulations. Drug-induced leftward shifts in ICSS frequency-rate curves are often interpreted as evidence of abuse liability. However, SNC80 (1.0-10 mg/kg s.c.; 10-56 mg/kg i.p.) failed to alter ICSS frequency-rate curves at doses up to those that produced convulsions in the present study or other effects (e.g. antidepressant effects) in previous studies. For comparison, the monoamine releaser d-amphetamine (0.1-1.0 mg/kg, i.p.) and the kappa agonist U69,593 (0.1-0.56 mg/kg, i.p.) produced dose-dependent leftward and rightward shifts, respectively, in ICSS frequency-rate curves, confirming the sensitivity of the procedure to drug effects. ICSS frequency-rate curves were also shifted by two non-pharmacological manipulations (reductions in stimulus intensity and increases in response requirement). Thus, SNC80 failed to facilitate or attenuate ICSS-maintained responding under conditions in which other pharmacological and non-pharmacological manipulations were effective. These results suggest that non-peptidic delta opioid receptor agonists have negligible abuse-related effects in rats.

The active site of deoxyhypusyl hydroxylase: use of catecholpeptides and their component chelator and peptide moieties as molecular probes.

The final step of hypusine formation in the eukaryotic translation initiation factor 4D (eIF-4D) is mediated by the enzyme deoxyhypusyl hydroxylase. In an effort to find specific inhibitors for this enzyme, we have studied the effects of two catecholpeptides, N alpha-acetyl-N delta-(3,4-dihydroxybenzoyl)-L-Orn-L-Pro-Gly (compound I) and N alpha-acetyl-N delta-(2,3-dihydroxybenzoyl)-L-Orn-L-Pro-Gly (compound II). Their structures were designed for anchorage to the enzyme s active site, utilizing the catechol-mediated chelation of a putative, enzyme-bound metal ion. Both compounds were found to strongly inhibit hypusine formation in vitro. Compound I was about seven times more potent than compound II, whereas the component peptide itself showed no intrinsic inhibitory activity even at concentrations as high as 1 mM. When used in conjugation with a chelating catechol moiety, however, it gave a 17- and an 8-fold enhancement of the half-maximal inhibition mediated by the chelating moieties per se, i.e. the 3,4- and the 2,3-dihydroxybenzoyl esters, respectively. The mode of inhibition by compound I was competitive with respect to the unhydroxylated precursor of eIF-4D and showed a Ki value of 32 microM +/- 3.4 microM. These catecholpeptides are the most efficient peptide antagonists of deoxyhypusyl hydroxylase known at present. They allow an assessment of the enzymes active site organization and provide the first experimental evidence that a metal ion constitutes an integral part of its catalytic center.