Pro-TRH-connecting peptides in the rat pancreas during ontogenesis

Rat thyrotropin-releasing hormone prohormone (pro-TRH) is a protein containing five copies of TRH, separated by connecting peptides. We have recently developed radioimmunoassays to synthetic peptides corresponding to prepro-TRH(160-169) and prepro-TRH(178-199). In the present study we have used these assays to investigate the ontogenesis of pro-TRH-derived peptides in the rat pancreas. Reverse-phase HPLC analysis of pancreatic extracts from 2-day-old rats showed the presence of two major immunoreactive peptides exhibiting the same retention time as synthetic prepro-TRH(160-169) and prepro-TRH(178-199), respectively. The concentrations of TRH and pro-TRH cryptic peptides in the rat pancreas rose rapidly after birth, reached a maximum at day 2-4 and decreased gradually afterwards. Streptozotocin treatment of newborn rats induced a marked decrease of TRH (96%), prepro-TRH(160-169) (97%) and prepro-TRH(178-199) content (94%) in pancreatic extracts. These results indicate that the evolution of TRH and pro-TRH-derived peptides follows the same pattern during the postnatal period. Our results also suggest that beta-cells are the only source of pro-TRH-derived peptides in the rat pancreas.     

Book reviews

Book reviewed in this article:
I ndustrial M icrobiological T esting (1987). Edited by J.W. Hopton & E.G. Hill.
D rinking W ater M icrobiology (1987). Edited by D.O. Cliver & R.A. Newman.
S urvival and D ormancy of M icroorganisms (1987). Edited by Y. Henis.
B rewing M icrobiology (1987). Edited by F.G. Priest & I. Campbell.
T he P athogenesis of B acterial I nfections (1985). Edited by G.G. Jackson & H. Thomas.
A dvances in M icrobial P hysiology Volume 28 (1986). Edited by A.M. Rose & D.W. Tempest.
A dvances in M icrobial P hysiology Volume 29 (1988). Edited by A.M. Rose & D.W. Tempest.
M icrobial Q uality A ssurance in P harmaceuticals C osmetics and T oiletries (1988). Edited by S.F. Bloomfield, R. Baird, R.E. Leak & R. Leech.     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.     

Disruption of disulfide bond formation alters the trafficking of prothyrotropin releasing hormone (proTRH)-derived peptides

Rat prothyrotropin releasing hormone (proTRH) is processed in the regulated secretory pathway (RSP) of neuroendocrine cells yielding five TRH peptides and several non-TRH peptides. It is not understood how these peptides are targeted to the RSP. We show here that a disulfide bond in the carboxy-terminus of proTRH plays an important role in the trafficking of this prohormone. Recombinant proTRH was observed to migrate faster on a native gel when treated with dithiothreitol (DTT) suggesting the presence of a disulfide bond. In vitro disulfide bond formation was prevented either by DTT treatment or by mutating cysteines 213 and 219 to glycines. In both cases the peptides derived from these mutants exhibited increased constitutive release and processing defects when expressed in AtT20 cells, a neuroendocrine cell line used in our prior studies on proTRH processing. Immunocytochemistry revealed that wild-type proTRH and mutant proTRH localized in a punctate pattern typical of proteins sorted to the regulated secretory pathway. These data suggest that the proposed disulfide bond of proTRH is involved in sorting of proTRH-derived peptides and in their retention within maturing secretory granules. This is the first evidence of structural motifs being important for the sorting of proTRH.     

Prothyrotropin-releasing hormone targets its processing products to different vesicles of the secretory pathway

Prothyrotropin-releasing hormone (pro-TRH) is initially cleaved by the prohormone convertase-1/3 (PC1/3) in the trans-Golgi network generating N- and C-terminal intermediate forms that are then packed into secretory vesicles. However, it is not known whether these peptides are differentially sorted within the secretory pathway. This is of key importance because the processing products of several prohormones fulfill different biological functions. Using AtT20 cells stably transfected with prepro-TRH cDNA, we found that two specific N- and C-terminal peptides were located in different vesicles. Furthermore, the C-terminal pro-TRH-derived peptides were more efficiently released in response to KCl and norepinephrine, a natural secretagogue of TRH. Similar sorting and secretion of N- and C-terminal peptides occurs in vivo. When we blocked the initial proteolytic processing by a mutagenic approach, the differential sorting and secretion of these peptides were prevented. In summary, our data show that pro-TRH-derived peptides are differentially sorted within the secretory pathway and that the initial cleavage in the trans-Golgi network is key to this process. This could be a common mechanism used by neuroendocrine cells to regulate independently the secretion of different bioactive peptides derived from the same gene product.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.     

Immunohistochemical localization of thyrotropin-releasing hormone (TRH) in skin of Rana pipiens

Summary Using immunofluorescent techniques thyrotropin releasing hormone (TRH) is demonstrated in skin of Rana pipiens and R. catesbeiana. The immunofluorescent-TRH is localized in all cell layers of the epidermis and in the epithelium lining the various cutaneous glands, but not in the dermal layer.We wish to thank Dr. Ronald DeLellis and Ms. Mary Blount for their expert advice and guidance in the immunohistochemical techniques.This investigation was supported by NIH National Research Service Award # 1F32 AMO6018-01 from the NIAMDD to Janice L. Bolaffi and NIH Grant AM 21863 to Ivor M.D. Jackson.     

Also received

Alcock , J. 1988. The Kookaburra's Song, Exploring Animal Behaviour in Australia. Atwood , J.L. 1988. Speciation and Geographic Variation in Black-tailed Gnatcatchers. Ornithological Monographs no. 42. Brazil , M. 1988. A Birdwatcher's Guide to Japan. Breslina , I.P. 1987. Plants and Waterfowl of Marine Islands in the Kola Subarctic. (Russian.) Brown , V., Weston , H. JR. & Buzzell , J. 1986. Handbook of Californian Birds. Darlington , D. 1987. In Condor Country. A Portrait of a Landscape, its Denizens, and its Defenders. Dunne , P., Sibley , D. & Sutton , C. 1988. Hawks in Flight. Gerrard J.M. & Bortolotti , G.R. 1988. The Bald Eagle; Haunts and Habits of a Wilderness Monarch. Hale , W.G. & Margham , J.P. 1988. Dictionary of Biology. Hovel , H. 1987. Check-list of the Birds of Israel. Ilyichev , V.D., Butiev , V.T. & Konstantinov , V.M. 1987. The Birds of Moscow and the Moscow Area. (Russian.) Ilyichev , V.D. & Fomin , V.E. 1988. The Fauna and Environmental Changes. (Russian.) In eight chapters, the authors attempt to describe and analyse the development (over about 200 years) of the present-day avifauna of the southern Urals, an area of considerable industrial and agricultural importance. Irisov , E.A. (ed.) 1987. Endangered, Rare and Little-known Plants and Animals of the Altai Territory and Associated Conservation Problems. (Russian.) Jennings , M.C., Salama , M.I. & Felemban , H.M. 1988. Report on an Ornithological Survey of the Asir National Park, Saudi Arabia, 29 June to 18 July 1987. Johnston , R.F. (ed.) 1988. Current Ornithology Volume 5. Lint , K.C. & Lint , A.M. 1988. Feeding Cage Birds. A Manual of Diets for Aviculture. Litvinenko , N.M. (ed.) 1987. Distribution and Biology of Seabirds of the Far East. (Russian.) Mineev , Yu . N. 1987. Waterfowl of the Bol ‘shezemel’ skaya Tundra. (Russian.) Ecological study of two Gaviidae and 20 Anatidae in the extreme northeast of the European USSR, an area bounded roughly by the Pechora river in the west and Vorkuta and the Kara river in the east. Pendleton , B.A.G., Millsap , B.A., Cline , K.W. & Bird , D.M. (eds) 1987. Raptor Management Techniques Manual. Porter , R.D., Jenkins , M.A. & Gaski , A.L. 1987. Working Bibliography of the Peregrine Falcon. Scientific & Technical Series no. 9. Pulich , W.M. 1988. The Birds of North Central Texas. The W.L. Moody Jr. Natural History Series no. 9. Robinson , J.W. 1987. A Birder's Guide to Japan Shvetsov , Yu .G. (ed.) 1988. Rare Terrestrial Vertebrates of Siberia. (Russian.) Silvius , M.J., Djuharsa , E., Taufix , A.W., Steeman , A.P.J.M. & Berczy , E.T. 1987. The Indonesian Wetland Inventory. Sitters , H.P. 1988. Tetrad Atlas of the Breeding Birds of Devon. Slater , P., Slater , P. & Slater , R. 1986. The Slater Field Guide to Australian Birds. Soper , T. with the British Trust for Ornithology. 1988. Go Birding. Soule , M.E. 1986. Conservation Biology. Steadman , D.W. & Zousmer , S. 1988. Galapagos. Telleria , J.L. (ed.) 1988. Invernada de Aves en la Peninsula Iberica. Monograph no. 1. Toups , J.A. & Jackson , J.A. 1987. Birds and Birding on the Mississippi Coast. Vasil'chenko , A.A. 1987. The Birds of Khamar-Daban. (Russian.) Wilbur , S.R. 1987. Birds of Baja California. Williams , L.E. & Austin , D.H. 1988. Studies of the Wild Turkey in Florida. Woolham , F. 1987. The Handbook of Aviculture. Zaboeva , I.V. (ed.) 1986. Distribution and Numbers of Animals in the European North. ?alakevi?ius , M. (ed.) 1987. Study of Nocturnal Autumn Bird Migration by the Electric Light of Hothouses. (Russian.) Zimmerman , J.L. & Patti , S.T. 1988. A Guide to Bird Finding in Kansas and Western Missouri.     

Multiple preprosomatostatin sorting signals mediate secretion via discrete cAMP- and tetradecanoylphorbolacetate-responsive pathways.

We have previously detected a sorting signal in the amino-terminal 78 residues of rat preprosomatostatin (rPPSS) that targets the precursor into a regulated secretory pathway or pathways allowing proteolytic maturation (Sevarino, K. A., Stork, P., Ventimiglia, R., Mandel, G., and Goodman, R. H. (1989) Cell 57, 11-19). To further localize this signal, we constructed three rPPSS expression vectors that code for substitutions or mutations spanning that portion of rPPSS implicated in sorting, and the precursors were expressed in RIN 5F cells. Fractionation of the intracellular products revealed that accurate processing to somatostatin-14 (SS-14) was not affected by any of the mutations. Examination of the secreted products showed no reduction in processing efficiency, indicating that none of the mutations blocked sorting from constitutive into regulated secretion. Finally, we examined the response to two separate secretogogues, cAMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Clones expressing two of the three mutant precursors displayed the same stimulation of SS-14 secretion by exogenously administered cAMP and TPA as cells expressing wild-type rPPSS, indicating that targeting specifically to the secretory pathway, or pathways, responsive to cAMP and TPA was not disrupted. However, cells expressing the mutant precursor containing a substitution of the amino-terminal 34 residues of rPPSS by the amino terminus of the vesicular stomatitis virus G protein displayed greatly reduced stimulation of SS-14 secretion by TPA, with a less than compensatory increase in response to cAMP, when compared to cells expressing wild-type rPPSS. In conjunction with our previous studies with anglerfish preprosomatostatins, we conclude that 1) the sorting signal(s) in rPPSS necessary for cAMP-responsive secretion are redundant and probably reside within both mature peptide regions and extrapeptide regions; 2) two or more distinct regulated secretory pathways utilized by secreted peptides can be demonstrated in transfected endocrine cells and targeting to these pathways can be separately mediated by at least two different types of sorting signals within the neuropeptide precursor itself; and 3) pro-region conformation plays little role in prosomatostatin-processing site recognition.     

Thyroid hormone modulation of TRH precursor levels in rat hypothalamus, pituitary, thyroid and blood

In the present study we have examined the in vivo effects of thyroid hormones and TRH on tissue and blood levels of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Using specific radioimmunoassays (RIAs), we measured TRH immunoreactivity (TRH-IR) and TRH-Gly-IR concentrations in blood, hypothalamus, anterior and posterior pituitary, and thyroid in euthyroid, hypothyroid and thyroxine (T4)-treated 250 g male Sprague-Dawley rats. TRH-Gly-IR and TRH-IR were detected in all of these tissues. Highly significant positive correlations between whole blood TRH-Gly-IR levels and the corresponding serum TSH values (p less than 0.01), whole blood TRH-IR versus serum TSH (p less than 0.01) and whole blood TRH-Gly-IR versus whole blood TRH-IR (p less than 0.01) are consistent with cosecretion of TRH and TRH precursor peptides into the circulation. Euthyroid rats injected with TRH IP (1 microgram/100 g b.wt.) and hypothyroid rats had 4-fold higher whole blood TRH-Gly-IR levels compared to euthyroid controls (p less than 0.0005). Injection of TRH into euthyroid rats significantly increased the TRH-Gly-IR concentration in the hypothalamus, anterior and posterior pituitary and thyroid. The increase in blood TRH-Gly-IR following intravenous TRH may be due, in part, to partial saturation of TRH-degrading enzymes in blood and cell membranes. The ratio of TRH-Gly to TRH was significantly increased in the anterior pituitary by hypothyroidism and TRH injection, suggesting that thyroid hormones and TRH regulate the alpha-amidation of TRH-Gly to form TRH in this tissue. TRH-Gly levels of pooled pituitary and thyroid extracts quantitated by a combination of TRH-Gly RIA and high performance liquid chromatography (HPLC) revealed several-fold increases following incubation at 60 degrees C. Heating at this temperature may block the alpha-amidation activity in extra-hypothalamic tissues but not the "trypsin-like" enzymes which cleave prepro-TRH into TRH-Gly-immunoreactive peptides.     

Inactivation of dopamine beta-hydroxylase by p-cresol: evidence for a second, minor site of covalent modification at tyrosine 357

p-Cresol is a mechanism-based inhibitor of bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate: oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) (DBH) which covalently modifies a tyrosine at position 216 during inactivation (DeWolf, W.E., Jr., Carr, S.A., Varrichio, A., Goodhart, P.J., Mentzer, M.A., Roberts, G.D., Southan, C., Dolle, R.E. and Kruse, L.I. (1988) Biochemistry 27, 9093-9101). Here we report the recovery and characterization of additional minor peptides that are produced during the inactivation of DBH with p-[3H]cresol. Sequence and structural analysis of these peptides indicates tyrosine 357 as a second, minor site of modification.     

Book reviews

Book reviewed in this article:
E lectrochemical D etection T echniques I n T he A pplied B iosciences Volume 2 (1988). By G-A. Junter
P lant R esistance T o V iruses (1987). Edited by D. Evered & S. Harnett
M icrobial R econtamination I n D airy P rocessing –P roceedings O f T he I nternational C ongress.
D evelopments I n F ood M icrobiology –4 (1988). Edited by R.K. Robinson.
C ellulose H ydrolysis (1987). Edited by L. T. Fan, M.M. Gharpuray & Y.-H. Lee
I mmobilization O f C ells (1988). By C.R. Phillips & Y.C. Poon
C rystalline B acterial C ell S urface L ayers (1988). Edited by U.B. Sleytr, P. Messner, D. Pum & M. Sara     

Books

Book reviewed in this article:
A ndrle , R.F. & C arroll , J.R. (eds) 1988. The Atlas of Breeding Birds in New York State.
B ergerud , A.T. & G ratson , M.W. (eds) 1988. Adaptive Strategies and Population Ecology of Northern Grouse.
C ooper , M.E. 1987. An Introduction to Animal Law.
G oriup , P.D. (ed.) 1988. Ecology and Conservation of Grassland Birds.
H udson , P.J. & R ands , M.R. 1988. Ecology and Management of Gamebirds.
J anssen , R.B. 1987. Birds in Minnesota.
J ones , P.H. 1988. The Natural History of Bardsey.
K ondratiev , A. YA. (ed.) 1988. Bulletin of the Working Group on Waders.
L ardelli , R. 1988. Atlante degli Uccelli Nidificanti net Mendrisiotto.
M arle , J.G. van & V oous , K.H. 1988. The Birds of Sumatra. B.O.U. Check-list No. 10.
P almer , R.S. (ed.) 1988. Handbook of North American Birds; Volumes 4 & 5: Diurnal Raptors.
R ose , M.R. 1987. Quantitative Ecological Theory: an Introduction to Basic Models.
S chleidt , V.M. (ed.) 1988. Der Kreis urn Konrad Lorenz.
S ick , H. 1985. Ornitologia Brasileira. 3rd edition. Vols I and II.
S ummers -S mith , J.D. 1988. The Sparrows.
S iokhin , V.D., C hernichko , I.I., A rdamatskaya , T.B. et al. 1988. Colonial Waterbirds in the Southern Ukraine: Charadriiformes.
T emple , S.A. & C ary , J.R. 1987. Wisconsin Birds: A Seasonal and Geographical Guide.
T rounson , D. & T rounson , M. 1987. Australia Land of Birds.
W oods , R.W. 1988. Guide to Birds of the Falkland Islands.     

The computer modelling of human TRH receptor, TRH and TRH-like peptides

The aim of this work was to verify the possibility of interactions between the human TRH receptor (an integral membrane protein which belongs to family 1 of G-protein coupled receptors) and TRH-like peptides presented in the prostate gland. These peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) for neutral or acidic amino acid, such as glutamic acid, phenylalanine, glutamine or tyrosine. The physiological function of TRH-like peptides in peripheral tissues is not precisely known. However, according to our recent experiments, we assume the existence of a local hormonal network formed by TRH-like peptides and TSH in the prostate gland. The network can be associated with circulating thyroid and steroid hormones, and may represent a new regulatory mechanism influencing the proliferative ability of prostatic tissue. A similar network of authentic TRH and TSH was already found in the gastrointestinal tract. The experimentally determined 3D-structures of human TRH receptor (hTRHr) and TRH-like peptides are not available. From this point of view we used de novo modeling procedures of G-protein coupled receptors on an automated protein modeling server used at the Glaxo Wellcome Experimental Research (Geneva, Switzerland). 3D-structures of TRH-like peptides were determined with a computer program CORINA (written by the team of J. Gasteiger, Computer-Chemie-Centrum and Institute for Organic Chemistry, University of Erlangen-Nurenberg, Germany). The generated PDB files with 3D-coordinates were visualized with Swiss-Pdb Viewer Release 3.51 (Glaxo Wellcome). From recent results it is evident that polar amino acids belonging to the extracellular terminus of hTRHr transmembrane regions can participate in interactions between TRH and hTRHr. There is no direct evidence that TRH-like peptides interact with the presented hTRHr model. On the contrary, with respect to the similar 3D-shape and the identity of terminal amino acids, it appears that these interactions are highly probable as well as the nearly 100 % cross-reactions between TRH or TRH-like peptides and antibody specific against authentic TRH. Closed terminal amino acids (pyroglutamic acid and proline-amide) of TRH or TRH-like peptides are important for these interactions. Desamido-TRH or glutamyl metabolites will be repelled by the negative potential of ASP195 (E: D93) and GLU298 (G: E137).     

Inactivation of dopamine β-hydroxylase by p-cresol: Evidence for a second,minor site of covalent modification at tyrosine 357

p-Cresol is a mechanism-based inhibitor of bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate; oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1) (DBH) which covalently modifies a tyrosine at position 216 during inactivation (DeWolf, W.E., Jr., Carr, S.A., Varrichio, A., Goodhart, P.J., Mentzer, M.A., Roberts, G.D., Southan, C., Dolle, R.E. and Kruse, L.I. (1988) Biochemistry 27, 9093–9101). Here we report the recovery and characterization of additional minor peptides that are produced during the inactivation of DBH with p-[³H]cresol. Sequence and structural analysis of these peptides indicates tyrosine 357 as a second, minor site of modification.     

Processing of TRH precursor peptides in rat brain and pituitary is zinc dependent.

The enzymes responsible for the posttranslational processing of precursor proteins to form alpha-amidated peptide hormones require the availability of several cofactors, including zinc, copper and ascorbate ions. Major changes in the availability of these cofactors, as well as the rate of hormone precursor conversion to active hormone, occur during neonatal development, aging and caloric restriction. The effects of 6 weeks of a zinc-deficient (ZD1) diet, pair feeding (PF) and partial zinc deficiency (ZD6) compared to a control diet on the enzymatic cleavage and processing of prepro-TRH to form TRH have been studied in the hypothalamus, brain, and pituitary of young adult male Sprague-Dawley rats. Reverse phase high pressure liquid chromatography (HPLC) revealed that TRH was the major TRH-IR component of the hypothalamus, brain and pituitary. The effect of zinc deficiency on the TRH-Gly-IR HPLC profile of rat brain was to reduce selectively the are of the peaks for TRH-Gly and other low molecular weight pro-TRH peptide fragments with a C-terminal Gly compared to the corresponding TRH-Gly-IR peaks of the control group. We conclude that the processing of prepro-TRH to form TRH is zinc dependent via posttranslational processing enzymes such as carboxypeptidase H.     

Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro- and neuropeptide Y

To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal.     

Evolutionary potential of (beta/alpha)8-barrels: stepwise evolution of a "new" reaction in the enolase superfamily

The molecular details of the processes involved in divergent evolution of "new" enzymatic functions are ill-defined. Likely starting points are either a progenitor promiscuous for the new reaction or a progenitor capable of catalyzing the new reaction following a single substitution that results from a single base change. However, the molecular (sequence) pathway by which the selective advantage provided by this protein can be improved and ultimately optimized is unclear. In the mechanistically diverse enolase superfamily, we discovered that a monofunctional progenitor could acquire the ability to catalyze a "new" reaction by a single base change: the D297G mutant of the monofunctional l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli catalyzed a low level of the o-succinylbenzoate synthase (OSBS) reaction as well as a reduced level of the AEE reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. We then discovered that the selective advantage and OSBS activity of the D297G mutant are both enhanced by the I19F substitution [Vick, J. E., Schmidt, D. M. Z., and Gerlt, J. A. (2005) Biochemistry 44, 11722-11729]. Both the D297G and I19F substitutions are positioned to alter the substrate specificity so that the substrate for the OSBS reaction is more productively positioned vis a vis the active site catalytic groups. We now report that both the selective advantage and OSBS activity of the D297G/I19F double mutant are enhanced by the R24C (one base change from the wild type Arg codon), R24W (two base changes from the wild type Arg codon and one base change from the R24C codon), and L277W (one base change from the wild type Leu codon) substitutions. The effects of the R24C and L277W mutants are "additive" in the D297G/I19F/R24C/L277W mutant. The greatest selective advantage and OSBS activity are associated with the D297G/I19F/R24W mutant. These "new" substitutions that enhance both the selective advantage and kinetic constants are positioned in the active site where they can alter the specificity, highlighting that the evolution of the "new" OSBS function can be accomplished by changes in substrate specificity.