Báo cáo khoa học: Biologically active, non membrane-anchored precursors – an overview ppt

Biologically active, non membrane-anchored precursors –an overview Eleni Dicou Institut de Pharmacologie Mole´culaire et Cellulaire du CNRS, UMR6097, Valbonne, France Introduction Precur

Biologically active, non membrane-anchored precursors –

an overview

Eleni Dicou

Institut de Pharmacologie Mole´culaire et Cellulaire du CNRS, UMR6097, Valbonne, France

Introduction

Precursor proteins mature through proteolytic cleavage

within the cell In most cases, these precursors are

bio-logically inert and their existence is limited to the

cyto-plasmic compartments where processing of secretory

proteins takes place

There are two sorting mechanisms in precursor⁄

pro-hormone secretion The first is the constitutive

path-way, in which newly synthesized proteins continuously

pass through the Golgi network and are

trans-ported in vesicles to the plasma membrane for

immedi-ate release The second is the regulimmedi-ated secretory

pathway, in which dense-core secretory granules that contain a condensed cargo of pro-hormones depend

on an extracellular stimulus for the release of the stored contents in a controlled manner This pathway

is operative in neuroendocrine cells and neurons Growth factors that derive from membrane-anchored precursors constitute an important exception

to this general model The membrane-anchored growth factor precursors are biologically active and, once they reach the cell surface, they can contact and activate cognate receptors on adjacent cells Thus, cleavage of their extracellular domain into soluble forms consti-tutes a process of conversion of one active form into

Keywords

bioactive precursors; chromogranins;

precerebellin; proapoA-I; proCHR;

proenkephalin; progastrin; proGRP;

proneurotrophins; PTH-P

Correspondence

E Dicou, Department of Biochemistry and

Molecular Biology, University of Texas

Medical Branch, Galveston, TX 77555-1072,

USA

Fax: +1 409 772 8028

Tel: +1 409 772 3686

E-mail: ln.dicou@utmb.edu

(Received 27 November 2007, revised 15

February 2008, accepted 28 February 2008)

doi:10.1111/j.1742-4658.2008.06366.x

Peptides function as chemical signals between cells of multicellular organ-isms via specific receptors on target cells Many hormones, neuromodula-tors and growth facneuromodula-tors are peptides Peptide hormones and other biologically active peptides are synthesized as higher molecular weight pre-cursor proteins (pro-hormones), which must undergo post-translational modification to yield the bioactive peptide(s) In many instances, more than one biologically active peptide is generated from one and the same precur-sor In most cases, these precursors are biologically inert and their existence

is confined to the membrane-enclosed subcellular compartments where pro-cessing of the pro-hormones takes place A class of growth factors that derive from membrane-anchored precursors which themselves are biologi-cally active constitute an exception to this model The list of the mem-brane-anchored biologically active precursors has been the subject of specialized reviews The present review focuses on precursors other than membrane-anchored precursors, which were found to be biologically active and which often display different biological activities, and may mediate their effects via receptors independent from those of their generated pep-tides

Abbreviations

ABCA1, ATP-binding cassette A1; ACTH, adrenocorticotrophic hormone; apo, apolipoprotein; BNDF, brain-derived neurotrophic factor; CCK 2

-R, cholecystokinin 2 receptor; Cg, chromogranin; CRH, corticotrophin-releasing hormone; GRP, gastrin-releasing peptide; HDL, high-density lipoprotein; IL, interleukin; LCAT, lecithin:cholesterol acetyltransferase; LPS, lipopolysaccharide; MNC, mononuclear cell; NGF, nerve growth factor; Penk, proenkephalin A; PPR, PTH ⁄ PTHrP receptor; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; SCLC, small cell lung carcinoma; TNF, tumor necrosis factor.

another rather than a process of pro-hormone

activa-tion The list of known membrane-anchored growth

factor precursors includes more than 10 members that

belong to the epidermal growth factor gene

super-family, precursors for tumor necrosis factor (TNF)-a,

colony-stimulating factor-1 and the c-kit receptor

ligand [1,2]

The present article provides an overview of the non

membrane-anchored, biologically active precursors,

which may have biological functions and act via

recep-tors that are distinct from those of their cleaved

pep-tides These include the precursor of cerebellin, the

family of chromogranins⁄ secretogranins,

proapolipo-protein (apo)A-I, procorticotrophin-releasing hormone,

progastrin, progastrin-releasing peptide, parathyroid hormone (PTH)-related protein, proenkephalin and the proneurotrophins (Fig 1) The present list includes only well-documented cases of biologically active precursors

Precerebellin

Precerebellin, Cbln1, is the prototype for a family of four brain-specific proteins (Cbln1–Cbln4) that was initially identified for harboring a naturally occurring 16-amino acid peptide, cerebellin [3] The peptide cere-bellin is abundant in Purkinje cells of the cerebellum and cartwheel neurons in the dorsal cochlear nucleus

Fig 1 Preprohormone amino acid sequences deduced from cDNAs h, human; m, mouse.

[4] During rat development, precerebellin mRNA

lev-els mirror the levlev-els of the cerebellin peptide Its levlev-els

increase in parallel with synapse formation during the

immediate postpartum period and decrease with

subse-quent synapse loss during remodelling In murine

mutants such as staggerer and weaver that have

per-turbed Purkinje cell synaptogenesis, cerebellin levels

are diminished

However, it has become increasingly apparent that

Cbln1 is not only a precursor, but also a signalling

molecule that is secreted from cerebellar granule cells,

which form synapses with Purkinje cells [3,5]

Electro-physiological and anatomical analyses of mutant mice

lacking the cbln1 gene have indicated that Cbln1 is

essential for synaptic integrity and plasticity in the

cer-ebellum and, in particular, in the matching and

main-tenance of pre- and postsynaptic structures and the

induction of long-term depression [5] Consequently,

cbln1-null mice display severe motor discoordination

and ataxic gait Interestingly, these abnormalities are

shared by mutant mice lacking the d2glutamate

recep-tor and it has been proposed that GluRd2 and Cbln1

may engage in a common signalling pathway crucial

for synapse integrity and plasticity

The cerebellin peptide is flanked by Val–Arg and

Glu–Pro residues Therefore, cerebellin is not

liber-ated from precerebellin by the classical dibasic amino

acid proteolytic-cleavage mechanism usually seen in

neuropeptide precursors The cerebellin peptide and

an N-terminal truncated version, des-Ser1-cerebellin,

are present in the cerebella from diverse vertebrate

species, suggesting that cerebellin is not a random

by-product of proteolysis Although abundant in the

cerebellum, cerebellin was also detected in the

hypo-thalamus, in ventromedial hypothalamic nuclei [6],

where it was implicated as a possible target of the

orphan nuclear receptor steroidogenic factor-1 and,

thus, may play a role in the development and⁄ or

migration of ventromedial hypothalamic neurons

Cerebellin was also shown to stimulate

norepineph-rine release and enhance adrenocortical steroid

secre-tion of the adrenal gland [7] It is found enriched in

synaptosomes and is released in a calcium-dependent

manner after depolarization, suggesting that it may

act as a neurotransmitter [8]

Although cerebellin has features of a neuropeptide,

the precursor Cbln1 belongs to the C1q⁄ TNF

super-family of secreted proteins, which suggests that it is

the biologically active molecule and that the

proteo-lytic events generating cerebellin serve another

func-tion Although precerebellin has no collagen motif, the

C-terminal two-thirds of the protein shows significant

similarity (52%) to the globular (noncollagen-like)

region of the B chain of human complement compo-nent C1q (gC1q) The gC1q signature domain, also found in many noncomplement proteins, has a com-pact jelly-role b-sandwitch fold similar to that of the multifunctional TNF ligand family [9] The members

of the ‘C1q⁄ TNF’ superfamily are involved in pro-cesses as diverse as host defense, inflammation, apop-tosis, autoimmunity, cell differentiation, organogenesis, hibernation and insulin-resistant obesity

Because most of the C1q signature domain proteins exist as an assembly of trimeric complexes, the exis-tence of a precerebellin family (Cbln1–Cbln4) was identified [10–13], suggesting that precerebellins are secreted proteins that function as heteromeric com-plexes Cbln1 was recently shown to form a trimer via its C-terminal C1q domain and a hexamer consisting

of two trimers connected via N-terminal disulfide bonds [14] Interestingly, cleavage at the N-terminus or C-terminus of the cerebellin peptide influences the state

of assembly of Cbln1 complexes [14] Each member has a C-terminal C1q domain and an overall amino acid sequence similarity with each other (60–80%) and they can form homomeric and heteromeric complexes

in mammalian cells in vitro [15] However, although the different Cbln subtypes are often coexpressed in certain brain regions, they have distinct patterns

of spatial and temporal expression in the adult and developing brain, indicating distinct roles for each member [13]

It is not yet known whether the cerebellin peptide or the precerebellins interact with specific receptors It is conceivable that the precerebellin complexes interact with a membrane receptor and activate an intracellular signal transduction cascade in a manner analogous to TNF-a

Chromogranins/secretogranins

The granin family comprises another example of pre-cursors that have biological activities distinct from their cleaved peptides The three classic granins are chromogranin (Cg)A, CgB and secretogranin II, in addition to four other less well known members, secre-togranins III–VI [16,17] The members of the granin family are uniquely acidic proteins ubiquitous in secre-tory cells of the nervous, endocrine and immune sys-tems They are proposed to play roles, first, in the formation and condensation of secretory granules by virtue of the ability of the granins to aggregate in the low pH, high calcium environment of the trans-Golgi network and, second, as a result of post-translational proteolytic processing, as pro-hormones that generate bioactive peptides

CgA has been proposed to act as an ‘on⁄ off’ switch

in the biogenesis of dense-core granules by a

mecha-nism involving upregulation of protease nexin-1, a

ser-ine protease inhibitor [18] Downregulation of CgA

using antisense RNAs in the PC-12 rat

pheochromo-cytoma cells leads to profound loss of dense-core

secretory granules and impaired secretion of

pro-opio-melanocortin Although transfection of CgA in a CgA

deficient PC-12 clone rescued the regulated secretory

phenotype, CgB expression was found to be important

in the induction of secretory granule formation in

non-endocrine CgB-transfected 3T3 and COS-7 cells [19]

Granins, besides their function in the biogenesis of

granule formation, also function as helper proteins in

the sorting of peptide precursors [20] or as inhibitors

of precursor processing [21] CgB, but not CgA, was

shown to have a nuclear localization in addition to its

localization in the cytoplasm and was implicated in a

transcription control role In gene array assays, CgB

induced or suppressed transcription of many genes,

including those of transcription factors [22]

Assays for granins, especially CgA, are of great

clin-ical use because circulating granins have served as

diagnostic markers for a variety of neuroendocrine

tumors [16] and in chronic heart failure [23] More

recently, two surprising functions were attributed to

CgA: the regulation of catecholamine-containing

dense-core chromaffin granule formation and the

con-trol of blood pressure in CgA knockout mice where

transgenic expression of the human CgA restored

blood pressure [24]

The presence of numerous paired basic amino acids

in granins suggests that they also give rise to peptides

as a result of post-translational proteolytic processing

Indeed, a variety of peptides derived from CgA, CgB

and other granin members have been identified and

shown to have autocrine, paracrine and endocrine

activities [25] Among them, vasostatins I and II

derived from CgA inhibit vasoconstriction, PTH

secre-tion, myocardial inotropy, vascular leakage and

micro-bial growth [17]; chromacin and catestatin, two other

fragments generated from CgA, as well as chrombacin

and secretolytin derived from CgB, also exert

bacterio-lytic and antifungal effects Pancreastatin from CgA

inhibits insulin release from pancreatic-islet beta cells

and modulates insulin responses in adipocytes and

hepatocytes whereas parastatin, containing the

catesta-tin region of CgA, also inhibits PTH secretion Other

granin-derived peptides are secretoneurin cleaved from

secretogranin II, which stimulates dopamine release

from nigrostriatal neurons, and 7B2 from

secretogra-nin V, which activates pro-hormone convertase PC2

[16,17]

Two interesting examples of precursor⁄ cleaved pep-tide opposing actions implicate vasostatin I and catest-atin CgA has anti-adhesive effects on fibroblasts and smooth muscle cells in vitro but its fragments (e.g after cleavage by plasmin) exert pro-adhesive effects [26,27] In hypertension, CgA is overexpressed whereas catestatin, a catecholamine release-inhibitory fragment,

is diminished via blocking of the nicotinic cholinergic receptor [24] Intraperitoneal injection of catestatin in CgA)⁄) mice resulted in the substantial reduction of their elevated blood pressure, analogous to the hista-mine-related hypotensive effect of intravenous injection

of catestatin in rats [28] However, to date, the recep-tors and⁄ or the mechanisms of action of CgA and its derived peptides remain elusive

ApoA-I

ApoA-I, the major protein of serum high-density lipo-protein (HDL), is a key element of the reverse choles-terol transport pathway, a process that removes cholesterol from extrahepatic tissues, including the ves-sel wall, thus protecting against the development of atherosclerosis [29] In this pathway, apoA-I defines the particle structure and stability of the HDL, pro-motes cholesterol efflux and activates lecithin:choles-terol acetyltransferase (LCAT) It is synthesized mainly

in hepatic and intestinal cells as a 267 amino acid pre-proprotein [30] The 18 amino acid leader sequence is cleaved during transit through the Golgi and a 249 amino acid proprotein is released into the plasma where the six amino acid propeptide (RHFWQQ) is proteolytically cleaved extracellularly to yield mature apoA-I The pro-segment of apoA-I is unusual in that

it terminates with a Gln-Gln dipeptide rather than a pair of basic amino acids Therefore, proapoA-I is itself the secretory form and proteolytic processing of proapoA-I to apoA-I occurs extracellularly

ProapoA-I is biologically active and, in several

in vitro studies, was shown to be functionally and structurally indistinguishable from mature apoA-I purified from plasma ProapoA-I secreted in a baculo-virus–insect cell system was found to bind lipid, and thus meet the essential criterion for its classification as

an apolipoprotein, and to stimulate LCAT activity as effectively as purified plasma apoA-I [31] However, using recombinant proapoA-I expressed in Escherichia coli, the ability of proapoA-I to bind to and reorganize phospholipid as compared to native apoA-I and the ability of the proform of apoA-I to form reconstituted HDL particles, as well as its capacity for LCAT acti-vation, were found to be very similar to the mature recombinant or native apoA-I forms [32] Although

most of newly secreted apoA-I and 3% of the plasma

apoA-I is proapoA-I, the biological function of

pro-apoA-I is not yet clear When the synthesis and

secre-tion of pro- and mature forms of apoA-I from a

baculovirus–insect cell expression system were

compared in parallel experiments, the amount of the

pro-form of apoA-I synthesized and secreted was

several-fold higher than that of the mature form of

apoA-I Furthermore, their ability to bind to plasma

HDL subfractions differed Twice as much proapoA-I

was found to be associated with preb1-HDL and

preb2-HDL subfractions compared to the mature form

but proapoA-I was found to be decreased in a1-HDL

and a2-HDL It is apparent, therefore, that the

pro-peptide is important for the effective synthesis and

secretion of apoA-I and that its deletion stimulates

conversion of preb-HDL to a-HDL [33]

A familial HDL deficiency, which is associated with

an increased risk of coronary heart disease, has been

characterized by reduced levels of apoA-I that were

not caused by reduced apoA-I production The

hyp-ercatabolism of the mature form, but not the

pro-form, was responsible for the HDL deficiency [34]

This comprises evidence to suggest that the pro- and

mature forms can be distinguished during HDL

metab-olism in vivo ProapoA-I has also been linked to

Tang-ier disease, a disease with abnormally low levels of

apoA-I and HDL In Tangier disease, proapoA-I is

present in approximately equivalent concentrations

compared to mature apoA-I and this is not due to a

deficiency of the converting enzyme activity [35] It is

thought that the differences in the levels of proapoA-I

versus apoA-I are a consequence of the rapid rate of

catabolism of apoA-I in Tangier disease due to its lack

of lipidation [36]

Other potential roles for the propeptide were

posed following the observation that deleting the

pro-peptide from preproapoA-I altered the efficiency of

in vitro cotranslational translocation⁄ processing, thus

suggesting that the propeptide plays a role in the

optimal folding of the precursor protein; it ‘helps’ the

nascent preprotein to assume an optimized

conforma-tion so that it may efficiently enter the secretory

apparatus [37] The propeptide also appears to play a

role in intracellular transport and to facilitate

trans-port of apoA-I out of the endoplasmic reticulum

[38]

The proapoA-I cleavage appears to be an

intermedi-ate step in the formation of biologically active preb1

-HDL Recently, the apoA-I proprotein convertase was

identified as the bone morphogenetic protein-1 and

shown to stimulate the conversion of newly secreted

proapoA-I to its phospholipid-binding form [39]

The mechanism of the formation of functional HDL from secreted lipid-free apoA-I has implicated the ATP-binding cassette A1 (ABCA1) transmembrane lipid transporter, which is responsible for the transfer

of phospholipid from cell membranes to circulating HDL [40,41] Notably, in Tangier disease, ABCA1 activity is congenitally deficient The absence of func-tional ABCA1 in Tangier disease, or its significant reduction in familial HDL deficiency patients, results

in the failure of newly synthesized apoA-I to acquire lipid, leading to rapid catabolism of lipid-poor nascent HDL particles [42]

The scavenger receptor type B class I was identified

as a high affinity HDL receptor that recognizes apoA-I Other receptors have also been postulated to be apoA-I

or HDL receptors, although the physiological relevance

of these findings remains to be established [30]

Procorticotrophin-releasing hormone

Corticotrophin-releasing hormone (CRH) is one of the main actors in the stress response in invertebrates and vertebrates [43] Studies mainly performed in mam-mals have demonstrated that CRH mediates the release of adrenocorticotrophic hormone (ACTH) from the pituitary, and this in turn leads to the release

of glucocorticoids from the adrenal gland CRH is a

41 amino acid peptide, produced as the C-terminal portion of a 196 amino acid CRH precursor (proCRH) After removal of the signal peptide and C-terminal amidation, this precursor, proCRH(27– 194), has a molecular mass of approximately 19 kDa ProCRH contains two potential cleavage sites, CS1(124–125) and CS2(151–152) Cleavage at CS2 would give rise to proCRH(27–151) and mature CRH whereas cleavage at CS1 would result in two other peptides: an N-terminal fragment proCRH(27–124) and the 8 kDa proCRH(125–151) ProCRH is expressed mainly in the hypothalamus and placenta

In the human normal term placenta, most of the CRH exists as unprocessed proCRH and pro-CRH(125–194) with very little in the form of CRH, except in pre-eclampsia, a disorder characterized by high blood pressure In the maternal plasma, CRH is the only one of the proCRH fragments to be main-tained in significant amounts in the maternal circula-tion [44]

ProCRH itself was shown to exert important biolog-ical effects Stably transfected CHO-K1 fibroblast cells expressing rat preproCRH synthesize and release the intact precursor, whereas no endoproteolytic products derived from proCRH were detectable in the extracel-lular medium ProCRH has a nuclear localization in

these transfected cells and appears to be in close

asso-ciation with DNA⁄ chromatin [45] ProCRH stimulated

the proliferation and DNA synthesis rate of the

trans-fected CHO-K1 cells compared to wild-type CHO-K1

cells Furthermore, treatment of mouse corticotrophic

tumor cells (AtT20⁄ D16-16) with conditioned medium

from transfected CHO-K1 cells expressing proCRH

stimulated both DNA synthesis and cell proliferation,

providing evidence of a mitogenic role for proCRH on

a corticotrophic cell population [45] ProCRH was also

effective in inducing ACTH release from primary

cul-tures of rat anterior pituitary cells, therefore acting as

an ACTH secretagogue in vivo [46]

ProCRH was also shown to be biologically active

within the immune system where it exerts an

immuno-modulatory action ProCRH, as well as CRH, has

been detected in human lymphocytes [47] ProCRH

exerted an inhibitory effect on basal and

lipopolysac-charide (LPS)-stimulated release of interleukin (IL)-6

by human peripheral blood mononuclear cells (MNCs)

[48] The dose of proCRH (nm range) effective for

inhibiting the release of IL-6 from MNC was the same

as that stimulating ACTH release from primary

cul-tures of rat anterior pituitary cells [46] This dose of

proCRH is also consistent with the dose of CRH

nor-mally used to stimulate ACTH release from

cortico-trophic cells, which further indicates a physiological

role for the intact precursor

It is interesting to note the opposing effects of

proCRH and CRH on IL-6 release from MNCs

ProCRH has an inhibitory effect whereas CRH

stimu-lates basal IL-6 release from MNCs [49] By contrast,

both have a stimulatory action, inducing ACTH

release from primary cultures of pituitary cells [46],

which suggests a dissociation between

immunoregula-tory and endocrine activities It has been suggested

that cellular components of the immune system may

be able to distinguish between closely related or

trun-cated peptides, whereas the classic neuroendocrine

tar-get cells might not [50]

A proCRH gene displaying a high degree of

homol-ogy with other proCRH genes known in vertebrates

has been isolated from the catfish Ameiurus nebulosus

[51] Interestingly, only one protein with a molecular

mass of 18 kDa, which is comparable to that of the

putative catfish proCRH peptide, was detected in all

tissues examined These results suggest that, in A

neb-ulosus, the proCRH does not require further

process-ing to be active and provide further evidence that

proCRH can exert itself important biological effects

Upon the stress response, besides activation of the

hypothalamic-pituitary-adrenal axis, the immune

sys-tem is also suggested to be actively involved A rapid

increase in proCRH levels was found in the central nervous system of the catfish A nebulosus after 15 min

of treatment with LPS [51] LPS is an immunologic challenger and could be considered as a stressor In this case, the increased proCRH could be a conse-quence of a response to LPS in which both immune and neuroendocrine systems are required for restoring body homeostasis [51] It is noteworthy that a close phylogenetic relationship and a high degree of conser-vation of proCRH and the CRH fragment is observed from invertebrates to vertebrates [52]

Progastrin

The hormone gastrin, first identified as a stimulant of gastric acid secretion [53], exists in two forms (17 and

34 amino acids, respectively), which share a common C-terminal sequence ending in an amidated phenylala-nine residue Both forms derive from a larger precur-sor molecule, the 101 amino acid preprogastrin, which

is rapidly converted to progastrin by cleavage of an

NH2-terminal signal peptide between residues 21 and

22 Amidated gastrin is believed to be the main biolog-ically active form, but recent studies have raised the possibility that non-amidated precursor forms of gas-trin, such as glycine extended gastrin (G-Gly) and progastrin, may also have growth factor properties [54]

Progastrin itself appears to act as a growth factor for normal colon, as transgenic mice expressing pro-gastrin in the liver have increased circulating concen-trations of progastrin and a hyperplastic colonic mucosa [55] Human colon cancers and colon cancer cell lines have been shown to express progastrin [56], and a possible autocrine growth factor role has been suggested, as in the case for gastrins [57] In addition, progastrin may act as a co-carcinogen in the develop-ment of colorectal carcinoma because, following treat-ment with azoxymethane, increased numbers of aberrant crypt foci and tumors were observed in the colonic mucosa of transgenic mice overexpressing progastrin compared to wild-type mice [58]

Recombinant human progastrin(1–80) stimulated proliferation and migration of the mouse gastric cell line IMGE-5 [59] Progastrin(1–80) was also shown to exert direct antiapoptotic effects on intestinal epithelial cells and upregulated cytochrome c oxidase [60] Under physiological conditions, only processed forms are present as the major circulating forms of gastrins in humans and rodents The full length pro-gastrin is generally not detected in the circulation In patients with colorectal cancers and hypergastrinemia, elevated levels of circulating progastrin were measured,

and it has been suggested that they may play a role in

colon carcinogenesis [56] Progastrin and G-Gly

repre-sent 90–100% of the gastrin peptides produced by

colon tumor and are found in 80–90% of colorectal

polyps in humans

Elevated levels of progastrin in the circulation of

transgenic mice overexpressing progastrin in the

intes-tinal mucosal cells resulted in significant alterations in

the emotional behaviour of these mice There was a

significant increase in the aggression, locomotor

activ-ity and anxiety-like behavior of the transgenic mice

compared to wild-type mice [61]

Amidated gastrins exert their effect through

activa-tion of their cognate receptors, cholecystokinin 2

receptors (CCK2-R) Low-affinity gastrin-binding sites

(Kd= 1.0 lm) termed CCKC-R bind progastrin and

gastrins [62] More recently, high affinity binding sites

were identified that were distinct from CCK2-R and

CCKC-R [63,64] The observations that recombinant

progastrin did not bind to the CCK2-R and that

antagonists to this receptor did not reverse the

prolif-erative effects of progastrin suggested that progastrin

stimulated proliferation independently of the CCK2-R,

probably via receptors specific to progastrin

Biologi-cally active recombinant human progastrin was found

to contain a tightly bound calcium ion and constitutes,

with the exception of proinsulin, comprising a first

example of selective, high affinity binding of metal ions

to a pro-hormone [63] More recently, annexin II was

identified as a high affinity progastrin binding protein

[65] A possible role of annexin II in mediating the

growth factor effects of progastrin was determined by

downregulating the expression of annexin II using an

antisense strategy

In response to progastrin, there is activation of Src

(which is an oncogene linked to colon cancer), the

phosphatidyl inositol 3¢-kinase ⁄ Akt pathway (which is

involved in the regulation of proliferation and

sur-vival), Janus-activated kinase 2, signal transducer and

activator of transcription 3 (which is recognized as an

oncogene implicated in many cancers) and

extracellu-lar-signal regulated kinases [66,67] Progastrin,

there-fore, is another example of a pro-hormone that is itself

biologically active and mediates effects via receptors

independent from those of its cleaved peptides

Progastrin-releasing peptide

Gastrin-releasing peptide (GRP) is a 27 amino acid

peptide with an amidated C-terminus and is a member

of the bombesin family of neuropeptides Bombesin

was originally isolated from the skin of the frog,

whereas GRP is the homologous peptide in mammals

It was initially characterized for its potent stimulation

of gastrin release [68] The widespread distribution of GRP, with significant amounts present in the central nervous system and throughout the gastrointestinal tract, suggests that it has more general actions It is now known to perform many other functions, includ-ing stimulation of the secretion of a variety of gastro-intestinal hormones and pancreatic enzymes, as well as the control of intestinal transit, smooth muscle con-tractility, metabolism and behaviour; it is also known

to regulate the immune system and to modulate smooth muscle contractility [69,70]

In particular, GRP has been recognized as the pro-totypical autocrine growth factor, based on the detec-tion of GRP and its cognate receptor in small cell lung carcinoma (SCLC) and on the anti-proliferative effect of GRP antibodies [71] GRP is also a potent mitogen for several other types of carcinomas, such

as colorectal, pancreas, prostate and breast tumors [72] GRP(1–27) is subsequently cleaved and amidated

to form GRP(18–27)

The precursor of GRP, proGRP, is a 125 amino acid protein and was shown to be biologically active [73] It was found to stimulate proliferation of the colon cancer cell line DLD-1 as efficiently as GRP(18– 27.) It also activates mitogen-activated protein kinase phosphorylation in these cells, as does GRP(18–27) This stimulation was reversed by the addition of an agonist of the GRP receptor, GRP-R, in the case of GRP, but not of proGRP Interestingly, proGRP dif-fered from GRP in that it failed to stimulate inositol production whereas GRP significantly stimulated inosi-tol production and this effect was reversed by the addi-tion of the GRP-R antagonist GRP mediates its effects via two receptors: the GRP-R and the BRS-3 receptors The proGRP appears to act through an independent receptor because, in binding experiments, proGRP did not compete with labelled bombesin for binding to GRP-R, nor did it compete with labeled BRS-3 agonist for binding to BRS-3 A GRP-R antag-onist blocked the effect of GRP, but not proGRP, on mitogen-activated protein kinase stimulation ProGRP was found to be present in several endometrial, pros-tate and colon cancer cell lines and in resected colorec-tal tumors [73]

GRP was expected to serve as a useful tumor mar-ker for SCLC patients; however, the instability of GRP in blood made its measurement difficult in clini-cal situations ProGRP (31–98), a region common to three isoforms of human proGRP, is stable in blood and can be conveniently measured by ELISA Neuron-specific enolase and carcinoembryonic antigen were also reported to be useful markers for patients with

SCLC However, proGRP was found to be superior in

terms of sensitivity [74] Assays for circulating proGRP

have also been used more recently as a tumor marker

for prostate and medullary thyroid cancer [75,76] The

possibility remains for using antibodies or antagonists

to proGRP in the treatment of colorectal and other

cancers that express proGRP Thus, proGRP is

another example of a pro-hormone giving rise to

bio-active peptides with independent receptors and

differ-ent bioactivities

PTH-related protein

Parathyroid hormone-related protein (PTHrP) has

been identified as an oncoprotein that is involved in

the pathogenesis of the paraneoplastic syndrome of

humoral hypercalcimia of malignancy It is structurally

related to PTH, the major regulator of calcium

homeo-stasis Unlike PTH, PTHrP does not circulate in

appreciable amounts in normal subjects but is

pro-duced by most cells and tissues in the body The

peri-natal lethality of PTHrP knockout mice emphasizes

the importance of this peptide system in normal life

Although PTHrP was discovered as a hypercalcemic

factor, one of its primary roles might be to regulate

differentiation, proliferation and death [77,78] The

dominant role of PTHrP as a developmental factor

has been well established in bone, skin and mammary

gland Such a role also appears to be relevant in most

other organs, including the cardiovascular system and

the kidney [79]

Following translation, PTHrP enters the secretory

pathway and, in cell types that possess the regulated

secretory pathway, such as pancreatic islet cells and

atrial cardiocytes, it is packaged into secretory

gran-ules and is subject to regulated secretion In tissues

that lack the regulated secretory pathway, such as

squamous carcinoma cells and fibroblasts, it is secreted

constitutively This duality of secretory mechanisms

indicates that PTHrP is unusual with respect to other

precursors in that it is both a neuroendocrine peptide

and a growth factor or cytokine During its transit

through the secretory pathway, the precursor is

endo-proteolytically processed at basic residues to yield a

family of mature secretory forms of the peptide [80]

PTHrP(1–36), displays smooth muscle relaxant

proper-ties and growth factor effects similar to PTHrP;

PTHrP(38–94⁄ 95 ⁄ 101) regulates calcium transport;

PTHrP(107–139), known as osteostatin, modulates

osteoclast activity; and PTHrP(141–173) stimulates the

growth of bone cells and collagen synthesis

Interest-ingly, the generated peptides may also have opposing

effects among themselves For example, PTHrP(1–36)

stimulates bone resorption whereas PTHrP(107–139) inhibits bone resorption

The best-studied biological effects of PTHrP are mediated through the binding of its NH2 terminus to a G-protein-coupled receptor, PTH⁄ PTHrP (PPR) that it shares with PTH [81] PPR signals through both the adenyl cyclase and phospholipase C second messenger pathways Pharmacological evidence supports the exis-tence of specific receptors for mid-region and carboxy-terminal PTHrP peptides; however, further research is required for their identification [81]

Recent studies have demonstrated that some of the biological actions of PTHrP are cell surface receptor independent and mediated through ‘intracrine’ mecha-nisms [77,82] The site between residues 87–107 of the PTHrP constitutes a nuclear⁄ nucleolar targeting sequence and is implicated in the role of PTHrP in cell cycle progression and apoptosis Such an intracrine mechanism has also been reported to increase cell pro-liferation This aspect raises new concepts in cellular protein trafficking However, the molecular mecha-nisms and the molecular targets of nuclear PTHrP remain unknown The PTHrP nuclear import appears

to be mediated by the transport receptor importin b [83] PPR has been detected in the nucleus in various cells and, hence, an active PTHrP⁄ PPR system may be functional at the nuclear compartment

Thus, in a single cell type, PTHrP may inhibit or stimulate proliferation or apoptosis, depending on whether it acts through the auto⁄ paracrine pathway or through the intracrine pathway [77,78,82]

Another role suggested for PTHrP might be related

to its nuclear localization PTHrP binds mRNA and this binding competes with a peptide corresponding to the nuclear⁄ nucleolar targeting sequence, implying that PTHrP may act as a nuclear export factor for mRNA [84] In a recent study, the role of PTHrP as an angio-genesis inhibitor on hair growth was proposed [85]

Proenkephalin A

Proenkephalin A (Penk) is one of the three opioid pre-cursor molecules (pro-opiomelanocortin, prodynor-phin, proenkephalin) which, upon complete processing

by cleavage at sites of dibasic residues, yield four cop-ies of the pentapeptide [Met]enkephalin, and one copy each of the pentapeptide [Leu]enkephalin, the hepta-peptide [Met]enkepalin–Arg6–Phe7 and the octapeptide [Met]enkephalin–Arg6–Gly7–Leu8 Enkephalins are naturally occurring peptides exhibiting opiate-like activity Enkephalins and opioid receptors have been identified in the brain, spinal cord, sympathetic ganglia and adrenal medulla, as well as in sympathetic and

parasympathetic neurons to the heart, spleen, vas

def-erens, stomach, intestine, lung, pancreas and liver [86]

Extended enkephalin-containing peptides that are

bio-logically active have been detected, as derived from

incomplete processing However, the biological

signifi-cance of Penk remained elusive for some time due to a

lack of appropriate antibodies because antibodies to

the small enkephalin peptides exhibited minimal or no

cross-reactivity with the full-length precursor The

sub-sequent generation of monoclonal antibodies to human

Penk-b-galactosidase fusion protein synthesized in

E colifacilitated the detection of the precursor [87]

The discrepancy between significant levels of Penk

mRNA but negligible amounts of mature enkephalin

peptides in bovine cerebellum [88] was confirmed using

monoclonal antibodies to the enkephalin precursor by

the immunofluorescent detection of Penk in

subpopu-lations of rat cerebellar neurons and in the absence of

mature enkephalin peptides [89] Penk was found to be

present at significant levels in astroglia cells [89,90] and

lymphocytes [91], and was released into the medium by

cultured astrocytes [92] These observations suggest a

biological role for Penk itself

A possible involvement of Penk in decision-making

events in growth control was demonstrated by its

nuclear localization in fibroblast and myoblast cells

[93] In cells that are in transition to growth arrest,

nuclear Penk responded promptly to mitogen

with-drawal and cell–cell contact by unmasking transiently

antigenic domains, which indicated the

acknowledg-ment of growth arrest and differentiation signals by

nuclear Penk

Opioids are known to affect survival and

prolifera-tion and their growth-promoting effects were found to

be mediated through Akt and Erk signalling cascades

[94] In addition, morphine has been shown to have

antitumor activity in vivo, mediated in part through

phosphorylation and activation of p53 [95] More

recently, Penk was implicated in apoptosis regulation

It was shown to physically associate with two

tran-scription factors: p53, known for its pro-apoptotic

function and its role as a tumor suppressor, and the

RelA(p65) subunit of nuclear factor-kappa B,

follow-ing UV-C irradiation and assistfollow-ing in apoptosis

through transcriptional repression of p-53 and nuclear

factor-kappa B gene targets [96] In addition, Penk

associates with high affinity to the transcriptional

co-repressor histone de-acetylase, which suggests that it

may be a component of a transcriptional repression

complex that contributes to a pro-apoptotic outcome

Penk, as well as the other opioid peptide precursors,

was shown to display sequence similarity with several

eukaryotic transcription factors [97]

A consensus regulated secretory pathway sorting sig-nal has been identified in Penk, which is similar to the sorting signal motif identified in pro-opiomelanocortin and proinsulin The mechanism involves the binding of the two acidic residues in the RSP sorting signal motif

to the two basic residues of the sorting receptor car-boxypeptidase E to effect sorting at the trans-Golgi network [98] Enkephalins interact with the d-opioid peptide receptors, although whether Penk interacts with the same receptors remains open to future investi-gation The availability of recombinant Penk should facilitate the search for other biological activities of Penk

Proneurotrophins

The neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4] are members of a family of homologous proteins that play

a critical role in the development, maintenance and regeneration of the nervous system These factors exist

in solution as noncovalently linked homodimers The biological effects of the neurotrophins are mediated by the Trk family of tyrosine kinase receptors (TrkA, TrkB, TrkC), and the low affinity receptor p75NTR, which is a member of the TNF receptor superfamily [99–101] Unlike the nonselective p75NTR receptor, which has a similar affinity for all neurotrophins, each Trk receptor selectively binds a different neurotrophin Neurtotrophins are initially synthesized as precur-sors that are subsequently proteolytically processed to release mature neurotrophin An NGF precursor form

of 31 kDa was initially detected in the rat thyroid [102], and NGF precursors of 31 kDa and 24 kDa were observed in the rat hippocampus [103] Following the initial observation that proNGF was the predomi-nant form in the rat thyroid with respect to NGF [102], it has been well documented that proNGF forms predominate in both central and peripheral tissues whereas the mature NGF peptide is rare [104] Several studies have suggested that the prodomain facilitated protein folding and promoted correct processing of biologically active NGF [105,106]

However, subsequently, proNGF and proBDNF were found to be secreted into conditioned media when they were expressed in heterologous cells [107–109], suggesting that they may act as ligands dis-tinct from the mature peptides Purified recombinant proNGF was shown to bind the p75NTR with higher affinity than NGF and to induce apoptosis [109] Later, it was found that proNGF binds simultaneously

to p75NTR and sortilin, a member of the Vps10p fam-ily of receptors, in a ternary complex Thus, sortilin

acts as a cell-surface coreceptor with p75 to mediate

proNGF induced cell death [110] ProBDNF also

induced neuronal apoptosis by binding to the

p75NTR⁄ sortilin complex, and proBDNF is secreted by

cultured neurons [111] Production of proNGF in vivo

by basal forebrain astrocytes was demonstrated after

kainic-acid induced seizures, indicating local

produc-tion of proneurotrophins under pathological condiproduc-tions

[112] Upregulation of proNGF and p75NTR after

spinal cord injury was shown to induce p75-mediated

death of oligodendrocytes, and proNGF present in the

injured spinal cord lysates induced apoptosis in culture

[113] Furthermore, using an axotomy model for the

induction of death of rat corticospinal neurons in vivo,

proNGF was shown to be secreted in the cerebrospinal

fluid of the lesioned animals and was capable of

trig-gering apoptosis in culture [114] Consequently, a

plau-sible role of proneurotrophins is to eliminate damaged

cells that express p75NTR

Thus, it is widely agreed that the Trk receptors

pro-mote cell survival and enhance synaptic transmission

upon binding of the mature neurotrophins; by

con-trast, the proneurotrophins preferentially bind to the

p75NTR⁄ sortilin complex to induce apoptosis This

dual system of ligand⁄ receptor assures neuronal fate

The duality of function of proneurotrophin⁄

neurotro-phin in the context of cell survival and death also

extends to the expression of plasticity in the brain

NGF and especially BDNF play important roles in

long-term potentiation via the Trk receptors In a

recent study, proBDNF was shown to enhance

hippo-campal long-term depression, whereas BDNF

facili-tates long-term potentiation [115]

Evidence that the pro-region may be important for

intracellular processing and secretion was provided in

a recent study of a single nucleotide polymorphism,

which converts a valine to methionine at codon 66

in the 5¢ pro-region of the human BDNF [116] This

substitution affected intracellular trafficking and

activity-dependent secretion of BDNF, leading to

impairment in hippocampal function Sortilin was

shown to interact specifically with BDNF in a region

encompassing the methionine substitution and to

control BDNF sorting to the regulated secretory

pathway [117] Interestingly, in another study, a

sort-ing motif within the mature BDNF was found to

interact with the sorting receptor carboxypeptidase E

and the substitution of two acidic residues with

ala-nine resulted in attenuation of the regulated secretion

of BDNF [118] Thus, elements present both in the

pro-region and the mature protein appear to control

the sorting of the BDNF to the regulated secretory

pathway

From the evidence provided above, it is clear that the precursors (proneurotrophins) and their generated peptides (neurotrophins) have a differential ability to bind to selective receptors and mediate distinctive bio-logical actions

Paradoxically, up to now, the processing of the proNGF and proNT-3 has been limited to the study

of the liberation of the NGF and NT-3 peptides How-ever, the NGF precursor sequence contains four sites

of dibasic amino acids and can yield two additional peptides of 29 amino acids (LIP1) and 38 amino acids (LIP2), whereas a 37 amino acid peptide can also be liberated from proNT-3 (elenin) ProBDNF cannot generate any other peptide except the BDNF

Chemically synthesized peptides that reproduce their sequences were shown to be biologically active They significantly inhibited the mitogenic activity of estro-gen, insulin-like growth factor and endothelial growth factor in MCF-7 breast cancer cells [119,120] LIP1 and LIP2 induced F-actin rearrangement and TrkA phosphorylation in PC-12 cells [121], which suggests that they mediate their action via the TrkA receptor, and enhanced cholinergic enzyme activities (choline acetyltransferase and acetylcholinesterase) in vivo in the cortex, septum and hippocampus of the neonatal hypothyroid rat [122] LIP1 and LIP2 bind and induce Akt phosphorylation in N11 microglial cells [119] LIP1 binds to sortilin with an approximately six-fold lower affinity than neurotensin, a ligand of sortilin, and thus may antagonize proNGF in certain cell con-ditions [119]

LIP1, LIP2, and elenin were neuroprotective against N-methyl-d-aspartate cytotoxicity in cultures of corti-cal neurons, and LIP1 and LIP2 also protected against ibotenate induced lesions in vivo [119] Furthermore, high levels of LIP1 and LIP2 were detected in the sera and synovial fluid of rheumatoid arthritis patients, sug-gesting that they are circulating peptides with a cyto-kine-like role [123] Thus, these peptides will further extend the list of the known members of the neurotro-phin family and again modify the known neurotroneurotro-phin family landscape

Conclusions

The present review has assembled information on ten biologically active precursors that are not membrane-anchored precursors All of the cited cases have been well-documented, and isolated reports of biologically active precursors for certain neuropeptides or hor-mones have not been included in this list Nonethe-less, this review does not claim to be an exhaustive list on the subject In general, from the above cited