MSH Activity Research Articles

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.

Overview of 45 Years of Studies on Peptide Chemistry

This review documents my research for the past 45 years in peptide chemistry. Initially, in order to study the structure-activity relationships of active center of alpha- and beta-melanocyte stimulating hormones (H-His-Phe-Arg-Trp-Gly-OH), we employed D-amino acids. That approach yielded first published report in 1965 of antagonists containing D-amino acids. Monkey beta-melanocyte stimulating hormone (beta-MSH), an 18 amino acid peptide stimulated pigment cells. We synthesized beta-MSH and fragments thereof, and studied in detail structure-activity relationships. A major and valuable result revealed that the C-terminal pentadecapeptide of beta-MSH exhibited higher MSH activity than the parent hormone providing a new question; namely, what was the role of the N-terminal tripeptide? In order to identify the novel enzyme, spleen fibrinolytic proteinase (SFP), I developed a specific chromogenic substrate, Suc-Ala-Tyr-Leu-Val-pNA, and a specific inhibitor, Suc-Tyr-D-Leu-D-Val-pNA, once again employing my D-amino acid strategy. SFP was purified by affinity chromatography using Suc-Tyr-D-Leu-D-Val-pNA as the bound ligand. The success of this approach provided me the incentive to develop a variety of potential drugs. Thus, I prepared a specific plasmin inhibitor (YO-2) and a plasma kallikrein inhibitor (PKSI-527). Next, my research developed novel opioid receptor specific opioid agonists and antagonists based on 2',6'-dimethyl-L-tyrosine (Dmt) dimers coupled with unique pyrazinone ring as a spacer. They exhibited potent oral antinociceptive activity acting through the mu-opioid receptor. Potent mu-receptor agonists (H-Dmt-Pro-Phe/Trp- Phe-NH(2)) were transformed into highly selective mu-receptor antagonists (N-allyl-Dmt-Pro-Phe/Trp-Phe-NH(2)), which reversed ethanol-induced increases in GABAergic neurotransmission, suggesting the possibility that they may emerge as candidates for the treatment of ethanol addiction.

The molecular genetics of the melanocortin pathway and energy homeostasis

The CNS melanocortin pathway plays an important role in the control of body weight. Two papers in this issue of Cell Metabolism, Lee et al., 2006 and Biebermann et al., 2006, suggest that beta MSH--a product of POMC processing--plays an unanticipated role in this pathway in humans.

Melanocortins and the Treatment of Nervous System Disease: Potential Relevance to the Skin?

For several decades melanocortins have been implicated in the modulation of brain function. More recently, this idea has been supported by the identification and cloning of melanocortin (MC) receptors in the nervous system. MCs stimulate axonal growth in fetal neural tissue or in neural cell lines in culture. This feature was utilized in screening their neurotrophic or neuroprotection potential in animal studies of nervous system disease (peripheral nerve and spinal cord trauma, toxic and metabolic neuropathies, EAN, EAE, etc.). Some of these effects may be mediated by MC4 receptor activation, although as yet unknown receptors may also be involved (for instance, protection by Org 2766). To what extent MC-nervous system effects are related to known effects of MCs in skin- and neuro-immune systems, remains to be discovered. Nevertheless, it is of interest to note that activation of brain MC4 receptors profoundly affects care behavior for the body surface (skin and fur). The excessive grooming response in rodents exhibits a remarkable functional correlation with MSH activity in a brain-skin axis.

Melanotropin as a potential regulator of pigment pattern formation in embryonic skin.

Frozen tissue sections of developing axolotl embryos were labeled by indirect immunofluorescence with anti-alpha-MSH. Anti-MSH immunoreactivity is first detectable in embryos when neural crest cells are migrating from the neural tube. Antibody labeling is visible around the lateral and ventral edges of the neural tube and in the embryonic ectoderm. As development progresses, the amount of labeling increases greatly, particularly in developing ectoderm. Western blots of soluble proteins extracted from various developmental stages of axolotl embryo ectoderm reveal that MSH activity is associated directly with several high molecular weight components that may be part of the embryonic extracellular matrix. Thus, we suggest that melanotropin activity is present in embryonic axolotl skin, is associated with the extracellular matrix, and is thereby in a position to play a supportive and/or directive role in the establishment of embryonic pigment patterns.

Estradiol and Plasminogen Activator Secretion by Cultured Rat Sertoli Cells in Response to Melanocyte‐Stimulating Hormones

Pro-opiomelanocortin-derived peptides, alpha-MSH and beta-endorphin, are synthesized and secreted by Leydig cells, and are believed to have paracrine effects on Sertoli cells in the testis. Peptides with MSH activity stimulate adenylate cyclase and cAMP accumulation in Sertoli cell-enriched cultures. The purpose of the present study was to determine whether such peptides would affect Sertoli cell parameters, such as aromatase and plasminogen activator activities, that are known to be regulated by cAMP. alpha-MSH stimulated aromatase activity in Sertoli cell-enriched cultures prepared from 10-day-old rats and this effect was potentiated by methyl isobutylxanthine (MIX). The combination of alpha-MSH plus MIX was not as potent as FSH. alpha-MSH, des-acetyl-alpha-MSH, beta-MSH, ACTH(1-13), and ACTH(1-24) stimulated aromatase activity to a similar extent, suggesting that Sertoli cells do not distinguish between the activities of these peptides. alpha-MSH potentiated the action of dbcAMP and forskolin on Sertoli cell aromatase, but unexpectedly had no effect on the action of either half-maximal or maximal doses of FSH. The regulation of plasminogen activator was examined next; urokinase was markedly suppressed by FSH in 10-day-old Sertoli cells. Although neither alpha-MSH nor MIX alone had an effect on urokinase secretion, in combination they were as effective as FSH. In 10-day-old Sertoli cells each of these peptides had little or no effect on tissue plasminogen activator.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of TRH on hormone release from pituitaries of the lizard, Anolis carolinensis

The ability of thyrotropin-releasing hormone (TRH) to stimulate thyrotropin (TSH) from pituitaries of adult male lizards, Anolis carolinensis, was tested in vivo and in vitro. TSH output by pituitaries in vitro was determined by bioassay of the incubation medium using in vitro T 4 output by thyroids from the same lizards. Pituitaries incubated without TRH had no detectable TSH secretion during two consecutive (2 or 3 hr) periods of incubation. Incubation in 10 or 100 ng/ml TRH for 2 or 3 hr significantly augmented release of TSH bioactivity in a dose-dependent manner. Pituitaries taken from goitrogen (100 μg methimazole/day for 10 days)-treated lizards had elevated basal TSH secretion but did not respond to TRH. TRH injection in vivo (5 μg/hr for 10 hr) appeared to stimulate acute release of MSH activity as judged by darkening of skin color after each injection, and plasma T 4 was significantly elevated at the end of treatment. These results provide additional evidence that the reptilian thyrotrope has functional TRH receptors and the TSH-stimulating activity of the tripeptide along with its effects on other pituitary cells was present at an early stage of reptilian evolution.

I. Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores

AspThrMetArg CysMetValGlyArgValTyrArgProCys TrpGluVal (melanin concentrating hormone, MCH) and several fragment analogs (MCH 1−14, MCH 5−17, MCH 5−14) were synthesized and their biological activities determined in a very sensitive fish skin bioassay. The potency ranking and minimum effective doses of the peptides were determined to be: MCH 1−17 (10 −12M) >< MCH 5−17 (10 −12M) > MCH 1−14 (10 −11M) > MCH 5−14 (2 × 10 −10M). The melanosome aggregating activity of MCH could be completely reversed by a 100-fold higher concentration of ≺-MSH. MCH was self-antagonized in a dose-related manner by higher concentrations of the peptide as was the activity of the MCH 1−14 fragment analog. The MCH activities of the MCH 5−17 and MCH 5−14 analogs were not compromised by even the highest concentrations of the peptides employed. The MSH-like activity of MCH appears to relate to the N-terminus of the peptide whereas MCH activity is more a function of the C-terminus of the hormone. Self- antagonism of MCH at high concentrations appears to relate to the N-terminal tetrapeptide, which is responsible for the intrinsic MSH-like activity of the hormone.

Differential structural requirements for the MSH and MCH activities of melanin concentrating hormone

▪ melanin concentrating hormone (MCH), exhibits both melanin granule concentrating and dispersing (MSH-like) activities. Fragment analogues of MCH were synthesized as described herein and the melanotropic activities of the peptides were determined. In the frog ( Rana pipiens ) and lizard ( Anolis carolinensis ) skin bioassays, the 5–17 and 5–14 fragments of MCH were inactive (at concentrations of 10 −5M or less), whereas the 1–14 sequence exhibited minimal (about 10%) MSH-like activity compared to MCH, which, as reported previously, was about 600 times less active than α-MSH. In the teleost (fish) skin bioassay, the MCH 5–17 analogue was equipotent to MCH, whereas the 1–14 analogue was 10–30 times and the cyclic N- and C- terminal truncated analogue, MCH 5–14, was about 300 times less active than MCH. These results suggest that the N-terminal sequence is particularly critical to MSH-like activity in the tetrapod species studied, whereas other structural regions of MCH, particularly in the C-terminal, are more related to MCH activity in teleosts.

Biochemical studies on pollen. Part XXXIII Purification and some properties of malate synthase from the pollen of Pinus densiflora Sieb. et Zucc.

Malate synthase (EC 4.1.3.2), termed MSH (mol. wt. 630, 000), was purified from the pollen of Pinus densiflora Sieb. et Zucc. to apparent homogeneity as judged by SDS-PAGE. Part of MSH was converted to the low molecular weight form of MS, termed MSL (mol. wt. 62, 000), by incubation with 5 mM ATP. Both forms of MS were maximally active at pH 7.6 in the presence of 10 mM MgCl2 and 0.01mM EDTA. MSL was completely inactivated by heating at 50°C for 3min, but MSH activity was retained about 65%. MSH preincubated with 5mM ATP showed unstability similar to that of MSL. MSH activity was more strongly inhibited than that of MSL by phosphoenolpyruvate or ATP. Km values of MSH for acetyl-CoA and glyoxylate were about one half of those of MSL. The Vmax of MSH was about 5 times as high as that of MSL. ATP was a noncompetitive inhibitor with respect to acetyl-CoA and Ki values of ATP for MSH and MSL were 1.0 × 10-3 M and 2.0 × 10-2 M, respectively. From these results we suggested that MSH and MSL are the active and low active forms of MS, respectively.

Effects of BAY K 8644 and Other Dihydropyridines on Basal and Potassium-Evoked Output of MSH from Mouse Melanotrophs in vitro

Dihydropyridines that have been shown in studies on other tissues either to facilitate Ca influx (BAY K 8644) or depress it (nimodipine and nifedipine) were examined for their effects on the secretory activity of the melanotrophs. Isolated mouse neurointermediate lobes, cultured for a week or longer to allow the nerves to degenerate, were perifused and output of MSH activity was measured by bioassay. The Ca-channel agonist BAY K 8644 augmented secretion under basal conditions and potentiated secretion evoked by 30 mM K+, a submaximally depolarizing concentration. The stimulant effect on secretion under basal conditions persisted in the presence of tetrodotoxin (which blocks the action potentials, Na spikes, in the melanotrophs) but was lost when Ca2+ was omitted or nimodipine added. The results are considered to support the view that the prominent basal secretory activity in these cells, as well as that evoked by excess K+, involve the inward flux of Ca2+ through dihydropyridine-sensitive Ca channels. If the stimulant and inhibitory effects of the dihydropyridines on basal secretion are to be attributed to actions on voltage-regulated Ca channels, as the results from other tissues would suggest, then such channels in the melanotrophs are different from those previously described in other tissues.

The Influence of the Nervous System upon Adrenal Gland Weight and Assayable Pituitary MSH

A series of experiments were designed to test the assumption that neuroregulatory mechanisms associated with pituitary ACTH and melanotropin (MSH) secretions are interdependent. Stress elevates both ACTH and MSH activity in mouse pituitaries. There are nervous influences on the secretion of both of these hormones. We have undertaken to stimulate or block various components of the nervous control of these 2 hormones in order to determine whether the 2 are controlled by the same mechanisms or separate ones. We have exposed mice to neurotransmitter blocking agents against serotonin, norepinephrine and epinephrin--parachlorophenylalanine (PCPA), alpha-methyltyrosine (MT), propranolol--both with and without stress. Unilateral adrenalectomy with and without hydrocortisone replacement therapy were investigated. We found that none of the neuro-active substances altered stress elevation of ACTH and MSH. Unilaterally adrenalectomized stressed animals treated with hydrocortisone showed elevated pituitary MSH levels but non increased ACTH release, as measured by size of the remaining adrenal. Adrenalectomy of animals without replacement therapy showed both elevated pituitary MSH and increased ACTH activity. Since the ACTH and MSH activity behave independently in the last described experiments, we conclude that ACTH and MSH are independently controlled.

Studies on peptides. LXXV. Synthesis of the hexadecapeptide corresponding to the entire amino acid sequence of .BETA.-melanocyte-stimulating hormone from the dogfish (Squalus acanthias).

In a conventional manner, the hexadecapeptide corresponding to the entire amino acid sequence of dogfish β-MSH (Squalus acanthias) was synthesized using protecting groups removable by hydrogen fluoride. The synthetic peptide exhibited the in vitro MSH activity of 1.88×109 MSH U/g.

ChemInform Abstract: STUDIES ON PEPTIDES. LXX. SYNTHESIS OF TWO OCTADECAPEPTIDES CORRESPONDING TO THE ENTIRE AMINO ACID SEQUENCE OF CAMEL β‐MELANOCYTE‐STIMULATING HORMONES

In a conventional manner, two octadecapeptides corresponding to the entire amino acid sequences of camel β1 and β2-MSHs were synthesized. Synthetic peptides exhibited the in vitro MSH activities of 5.1×109 and 1.1×109 MSH unit/g, respectively.

Studies on peptides. 70. Synthesis of two octadecapeptides corresponding to the entire amino acid sequence of camel .BETA.-melanocyte-stimulating hormones.

In a conventional manner, two octadecapeptides corresponding to the entire amino acid sequences of camel β1 and β2-MSHs were synthesized. Synthetic peptides exhibited the in vitro MSH activities of 5.1×109 and 1.1×109 MSH unit/g, respectively.

The immunolocalization of ACTH and α MSH in human and rat pituitaries

Human and rat pituitaries were investigated immunohistochemically for ACTH and alpha MSH activity by means of the enzyme-labeling technique. In rat pituitaries cells present in both the anterior and intermediate lobes were reactive with the anti-ACTH antibodies, the cells from the intermediate lobe were also reactive with anti-alpha MSH antibodies. In human pituitaries, ACTH-immunoreactivity was found in cells from the anterior lobe and cells invading the posterior lobe. In 5 out of 15 pituitaries ACTH-immunoreactive cells located at or invading the posterior lobe were also reactive with the anti-alpha MSH antibodies. It is concluded that the human pituitary cells that invade the posterior lobe represent a population which is at least immunohistochemically identical with the intermediate lobe cells of the rat. The ACTH-immunoreactivity of intermediate lobe cells may be explained by the presence of a corticotropin-like intermediate lobe peptide (CLIP) which has been suggested to be a prohormonal fragment of alpha MSH.

Lack of Detectable Secretion of ACTH from Pituitary Homografts of Pars Intermedia

Hypophysectomized rats bearing grafts of the pars intermedia (PI) in the kidney capsule for 20 days did not show adrenal weights significantly different from those of hypophysectomized controls. Plasma corticosterone was undetectable in the grafted rats, even after the injection of lysine-vasopressin or histamine. On the other hand, MSH activity was present in measueable amounts, independent of the drug administered, in the plasma of the grafted rats. These results suggest that PI transplants do not have the ability to release ACTH, even though it has been previously reported that they contain this hormone.

DEVELOPMENT OF A RADIOIMMUNOASSAY FOR α-MELANOCYTE-STIMULATING HORMONE IN THE RAT

SUMMARY A sensitive and specific radioimmunoassay for α-melanocyte-stimulating hormone (α-MSH) was developed. Extracts of the neurointermediate lobe of the rat produced displacement curves which were parallel to those obtained with synthetic α-MSH. The mean immunoreactive a-MSH concentration in neurointermediate lobes from normal adult rats was 2768 ± 200 (s.e.m.) ng/lobe. This accounted for approximately 78% of the MSH activity of the neurointermediate lobe as measured by bioassay. Much lower levels of immunoreactive α-MSH were found in the anterior lobe of the rat. Extracts of rat serum and plasma also contained immunoreactive α-MSH and the mean level was found to be 237 ± 20 pg/ml. This was slightly lower than the level measured in rat plasma by bioassay. Increased levels of α-MSH were found in plasma of rats 1 and 3 h after a single injection of trifluoperazine and after 1·5 min of ether anaesthesia. These changes were reflected by decreases in the α-MSH content of the neurointermediate lobe.

Studies on peptides. LVII. Synthesis of the octadecapeptide corresponding to the entire amino acid sequence of equine beta-melanocyte-stimulating hormone.

The octadecapeptide corresponding to the entire amino acid sequence of equine β-melanocyte-stimulating hormone (MSH) was synthesized by assembling 4 subunits ; Z (OMe)-Asp (OBzl)-Glu (OBzl)-Gly-OH (I), Z (OMe)-Pro-Tyr-Lys (Z)-Met-NHNH2 (II), Z (OMe)-Glu (OBzl)-His-Phe-Arg (Tos)-Trp-Gly-OH (III), Z (OMe)-Ser-Pro-Arg (Tos)-Lys(Z)-Asp (OBzl)2 (IV), followed by deblocking of all protecting groups with hydrogen fluoride. The in vivo MSH activity of synthetic equine β-MSH was found equivalent to that of synthetic human β-MSH (in vitro activity, 6.2×109 MSH unit/g).

Studies on peptides. XLIX. Synthesis of the octadecapeptide corresponding to the entire amino acid sequence of porcine beta-melanocyte-stimulating hormone.

The octadecapeptide corresponding to the entire amino acid sequence of equine β-melanocyte-stimulating hormone (MSH) was synthesized by assembling 4 subunits ; Z (OMe)-Asp (OBzl)-Glu (OBzl)-Gly-OH (I), Z (OMe)-Pro-Tyr-Lys (Z)-Met-NHNH2 (II), Z (OMe)-Glu (OBzl)-His-Phe-Arg (Tos)-Trp-Gly-OH (III), Z (OMe)-Ser-Pro-Arg (Tos)-Lys(Z)-Asp (OBzl)2 (IV), followed by deblocking of all protecting groups with hydrogen fluoride. The in vivo MSH activity of synthetic equine β-MSH was found equivalent to that of synthetic human β-MSH (in vitro activity, 6.2×109 MSH unit/g).