JYI Volume Four Features: A Role For Proteolysis During Embryonic Development

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Volume Four

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Issue 2, August 2001

A Role For Proteolysis During Embryonic Development

Adam Friedman
Princeton University
friedman@jyi.org

Introduction

The proper growth, differentiation, and movement of cells during embryonic development depend upon the correct temporal and spatial organization of molecular cues given by and in relation to other cells in the embryo. These cues may take the form of gradients of secreted molecules, direct cell-cell interactions, or asymmetric localization of intracellular determinants. One major challenge for developmental biologists is to determine how these signals are generated and how the cell receives and integrates this information into a response. A simple observation of the end result, i.e., the adult animal, indicates the precision and complexity of this problem.

Convergence of several decades of research has revealed that organisms utilize only a few signaling pathways throughout embryonic development. However, these common pathways are tailored to a particular spatial and temporal context by modulating factors. One such factor is the transcriptional control of genes through specific regulatory regions. For example, in Drosophila melanogaster, the gene decapentaplegic (dpp) is required at multiple steps during early embryonic development (dorsal specification and dorsal closure) and later development of the wing from larval imaginal discs; its genomic structure consists of over 40 kilobases, most of which are cis-regulatory regions (Padgett et al. 1987). Other sources of control over the temporal and spatial patterning include post-transcriptional controls over RNA localization (e.g., nanos; Gavis and Lehmann 1994) and activation (e.g., polyadenylation; Salles et al. 1994), or post-translational control via sequestering (e.g. cactus; Roth et al. 1991), or by proteolysis of signal precursors (e.g., BMPs; Roebroek et al.. 1998). This review addresses the control over spatial and temporal localization of protein activity by means of this latter mechanism, the proteolytic activation of secreted factors.

Introduction to Proteolysis

Many proteins are translated as larger inactive precursors. During blood clotting, for example, a cascade of serine proteases are secreted as inactive zymogens which are successively cleaved, leading to cross-linkage of fibrin in the clot scaffold. Here, successive activation of multiple proteases allows for rapid amplification of a single trauma signal (Garrett and Grisham 1999). One of the earliest indications of the significance of proteolytic activation of precursors came from the observation than many hormones were synthesized intracellularly as prohormones (Nakayama 1997). Most of these precursors contain an N-terminal signal sequence ("Pre") which directs the translation product to the appropriate subcellular compartment, and a intervening "Pro" domain before the active protein. Families of proteases include relatively non-specific proteases such as trypsin, which cleave proteins after particular amino acids; metalloproteases which utilize metal ions such as zinc; and caspases, which are involved in apoptosis. An additional category of serine proteases and the subject of this review is the relatively sequence-specific, subtilisin-like, calcium-dependent serine protein convertase (SPC; Garrett and Grisham 1999; Neurath 1999).

The founding member of the convertase class of proteases is subtilisin, a bacterial protease (Figure 1), to which all SPCs share structural similarity. The importance of convertases in higher species was realized after the discovery that the Saccharomyces cerevisiae homologue of subtilisn, Kex2p (kexin), which is required for the maturation of the yeast a-mating factor, could process human proalbumin and prepro-piomelanocortin (mPOMC) (Nakayama 1997; Chrétien et al. 1995; Bathurst et al. 1987). Subsequently, more than ten KEX2-like genes have been identified in the mouse, frog, worm, and fly combined (Chrétien et al. 1995; Roebroek et al. 1991; Hayflick et al. 1992). These include the mouse genes Furin/SPC1, SPC2, SPC3, SPC4/PACE4, SPC6A, SPC6B, and SPC7; the known fly genes dFurin-1 (dKLIP), dFurin-2, and amontillado (amon), and the known Xenopus laevis and Caenorhabditis elegans genes Xen-14 and bli-4, respectively.

Considerable research has been directed at understanding the evolution and significance of the "Pro" domain and the necessity for its cleavage by SPCs. For developmentally significant molecules, the presence of the "Pro" domain appears to permit regulated local activation of morphogens (e.g. Constam and Robertson 1999; Roebroek et al. 1998), while for other proteins, the "Pro" domain may serve to regulate intracellular stability or provide kinetic advantages during protein folding (Cunningham et al. 1999). Convertases cleave proproteins' C-terminal to a conserved consensus dibasic site within the Pro-domain (-R-X-X-R-, where "X" is any intervening amino acid) with few exceptions (Nakayama 1997). As with all serine proteases, this cleavage is accomplished through a "catalytic triad" of conserved histidine, aspartic acid, and serine residues (Garrett and Grisham 1999).

Molecular Characterization of SPCs

Convertases are remarkably similar both between and within species in their sequence similarity, maturation, subcellular localization, and substrate specificity, but have distinct mechanisms of transcriptional control and differential expression patterns in adult tissues. SPCs are highly conserved (50-70% amino acid identity), even outside of their catalytic domain. Moreover, Furin, SPC6A/B, dFur1, dFur2, and SPC7 all contain a C-terminal transmembrane domain as well. All SPCs are secreted as zymogens that are activated by autoproteolysis or by other convertases in the endoplasmic reticulum and Golgi apparatus (Nakayama 1997). Many permanently reside primarily in the trans-Golgi network (TGN), where they then process their targets passing through the secretory pathway. All of the convertases appear to have overlapping substrate specificities for the consensus Furin cleavage site, and therefore several may be involved with the in vivo cleavage of a particular substrate, or each may be required at a different time or place (Creemers et al. 1993; Nakayama 1997).

The regulation of convertase expression in adult tissues is complex and not well understood. Although most are expressed as multiple transcripts generated either by differential splicing or from several promoters, only a few are known to have more than one enzyme isoform (De Bie et al. 1995). For example, Furin is transcribed from at least three different promoters (Ayoubi et al. 1994) and all transcripts appear to be nearly uniformly distributed in all tissues of the body (Nakayama 1997; Chretien et al. 1995). SPC2 and SPC3, however, are expressed primarily in neuroendocrine tissues such as pancreatic islets and the pituitary (Constam and Robertson 1996; Nakayama 1997; Chrétien et al. 1995). Drosophila Furin-1 is expressed as four different transcripts encoding variations of the Dfurin-1 protein C-terminal to the catalytic domain; these transcripts are expressed in a non-overlapping pattern of tissues primarily in the central nervous system, fat bodies, and oviduct (Roebroek et al. 1993). The Dfurin-2 gene generates a single transcript; its adult expression pattern has not been reported (Roebroek et al. 1992, Roebroek et al. 1995).

Protein convertases have been implicated in a number of human pathophysiologies. The 250-residue precursor prepro-opiomelanocortin (POMC) is sequentially cleaved by SPC1/2 into an entire family of hormones, including an endorphin, adrenocorticotrophic hormone (ACTH), and melanocyte-stimulating hormones (Garrett and Grisham 1999). A rare form of ACTH deficiency has been traced to a congenital defect in SPC1 cleavage of the ACTH prohormone. In addition, some cases of familial hyperproinsulinemia have been traced to point mutations in the processing site of the pro-insulin precursor (Chrétien et al. 1995). Because of their role in human diseases, convertases have been identified as pharmacologic targets for treating diseases caused by abnormal secretion of proteins. The requirement of many viruses, such as the human immunodeficiency virus (HIV), to cleave coat proteins such as gp160 before cell entry, and the processing of procytotoxins such as diptheria toxin, have led to the development of a class of convertase inhibitors that are used as pharmacological agents (Chrétien et al. 1995; Jean et al. 1998; Nakayama 1997).

Proteolytic Processing in Embryonic Development

The importance of proteolysis in the regulation of developmental processes has been studied intensively, but only recently. Proteolysis appears to be a method of post-translational control over the spatial and temporal specificity of developmental molecular pathways, as well as serving a general housekeeping function (Jones et al. 1996). One of the most extensive examples of the significance of localized protein processing is the establishment of the Drosophila dorsal-ventral axis. In response to a ventralizing signal from the gurken-torpedo pathway, a cascade of inactive serine proteases including Gastrulation Defective, Snake, and Easter are sequentially processed in the perivitelline space, leading to localized processing of the Spätzle protein on the presumptive ventral side of the embryo. Spätzle, in turn, activates the Toll receptor, which causes localized nuclear accumulation of the Dorsal protein (Han et al., 2000; Morisato and Anderson 1994; Morisato and Anderson 1995). Here, ubiquitous molecules are locally activated by proprotein processing, in a process reminiscent of the blood clotting cascade.

Other well known signaling molecules have also been implicated as targets of proteolysis. Hedgehog (Hh), which contains some structural similarity to serine proteases, has been shown to undergo autoproteolysis, and the two cleavage products have been suggested to be responsible for the short-range and long-range signaling capacity of Hh (Lee et al. 1994). The proteolysis of the Notch receptor, a cell-cell signaling molecule, has been studied extensively. At least three cleavage events are required for the activity of Notch: (i) an extra-cellular domain is first cleaved by a furin-like convertase in the secretory pathway, and is placed as a heterodimer of its N-terminal and C-terminal halves in the plasma membrane (Blaumueller et al. 1997; Logeat et al. 1998); (ii) upon ligand binding, Notch can be cleaved extracellularly, and (iii) a third, presenilin-dependent cleavage releases the intracellular domain from the cell membrane (Mumm et al., 2000; Huppert et al., 2000; Struhl and Greenwald 1999). This intracellular domain then functions as a transcription factor in concert with the CBF1/Suppressor of Hairless [Su(H)]/Lag1 (CSL) class of transcription factors. The significance of the intracellular cleavage has been shown by generating mice homozygous for a processing-site mutation; these mice phenocopy Notch null mutants in their overall developmental delay, angiogenesis defects, and embryonic lethality (Huppert et al., 2000).

The developmental significance of SPCs has only recently been investigated. However, the role of the Drosophila convertases, Dfur-1, Dfur-2, and Amontillado is poorly understood, and their targets are not known. Understanding the embryonic expression patterns of these genes has not been helpful in identifying the Drosophila convertase targets. Both Dfur-1 and Dfur-2 appear to be supplied maternally and ubiquitously at high levels, but their transcripts mostly disappear early in development. For Dfur-1, the various isoforms (see above) are expressed in non-overlapping patterns shortly thereafter in tissues primarily in the central nervous system; Dfur-2 expression is not detected early in development, but appears in the central nervous system as well, in a pattern that does not overlap with Dfur-1 (Hayflick et al. 1992; Roebroek et al. 1995; Roebroek et al. 1993). Only one Dfurin-1 mutant line exists (Spradling et al. 1999), and, although it is known to be embryonic lethal, its phenotype has not been characterized. Amontillado (amon) was isolated in a PCR screen based on its striking similarity to SPC2 (81% amino acid similarity in its subtilisin-like catalytic domain). Mice homozygous for a deletion of SPC2, which is expressed primarily in neuroendocrine cells, are defective in prohormone processing and have altered pancreatic islet morphology (Furuta et al. 1997; Furuta et al. 1998). Amon-deficient fly larvae fail to leave their egg cases during hatching, most likely due to reduced thrashing. Amon is therefore implicated in the processing of Drosophila proneuropeptides and prohormones necessary for hatching (Siekhaus and Fuller 1999). As expected, amon expression peaks sharply before hatching and reduces shortly thereafter (Hwang et al., 2000; Siekhaus and Fuller 1999).

Mammalian SPC processing of TGFb homologues during development

Studies of SPC proteases during mammalian development have focused on their role in regulating the transforming growth factor b (TGFb) class of signaling molecules. The superfamily of TGFb molecules has been implicated in a wide range of cellular events, including proliferation, changes in cell shape, apoptosis, and specification of cell fate, leading to early axial patterning, organogenesis, and tissue homeostasis (Raftery and Sutherland 1999). Cells can adopt different fates with as little as 1.5-fold changes in concentration of TGFb signals (Constam and Robertson 1999). Examples from the TGFb class of signaling molecules have been found in sea urchins, nematodes, frogs, flies (e.g., decapentaplegic, screw, and glass-bottom-boat), and vertebrates (e.g. Bone Morphogenetic Proteins, or BMPs; Raftery and Sutherland 1999; Hogan et al. 1994). The importance of BMPs in mammalian development is illustrated by the wide range of developmental defects in BMP knockout mice, from ventral closure defects (BMP1; Suzuki et al. 1996) to severe early mesodermal defects (BMP4; Winnier et al. 1995) to mild bone defects (BMP6; Solloway et al. 1998).

Because of their wide range of effects and their evolutionary conservation, TGFb family members have been extensively studied, but the role of processing in regulating their activity has only been appreciated within the last decade. All TGFb proteins are synthesized as proproteins (Table 1). Processing at their conserved furin processing site RXXR releases the active C-terminal end (Hogan et al. 1994). The TGFb proprotein appears to be stabilized by the presence of the Pro domain; even after cleavage, the Pro domain can associate with the mature product to supply further stability to the active molecule (Constam and Robertson 1999).

One of the most striking demonstrations of the requirement of TGFb processing has been in studies of Vg-1, a mesoderm-inducing molecule in Xenopus. Injection of Vg-1 RNA has no effect on Xenopus embryos, while fusion of Vg-1 to mouse BMP4 or BMP2 Pro-regions induces dorsal mesoderm. The injected Vg-1 alone was not processed and thus not active, leading to the conclusion that regulated processing of Vg-1 may be important for its developmental control (Jones et al. 1996).

The expression patterns of several proprotein convertases during mouse embryonic development suggest that proteolytic processing is a mechanism for TGFb regulation. Furin/SPC1 and SPC7 appear to be expressed ubiquitously throughout the developing and adult mouse, suggesting that their activities are primarily constitutive or housekeeping. In contrast, SPC4/PACE4 and SPC6 are expressed in specific domains during embryogenesis: between embryonic days 8.5 and 11.5, SPC4 transcripts are detected in specific regions of the developing heart, neural tube, and the limb apical ectodermal ridge (AER), while SPC6 is detected primarily in the somites and the AER; at later stages, SPC4 is upregulated in the developing bones and, along with SPC6, the gut. The expression of SPC4 and SPC6 in the AER overlaps with that of BMP2, BMP4, and BMP7; SPC4 and BMP6 expression overlap in the floor plate and possibly in the chondrocytes. It was suggested that the specific expression of particular convertases such as SPC4 and SPC6 may serve to amplify the constitutive processing of the ubiquitous convertases furin and SPC7, thus locally enhancing the BMP inducing signal (Constam and Robertson 1996).

Several subsequent studies have strengthened the idea that convertases process BMPs during mouse development. Not surprisingly for proproteins with the conserved furin processing site, furin and SPC4 have been shown to process BMP4 in vitro (Constam and Robertson 1999). Injection of a furin-specific peptide inhibitor phenocopies BMP-4 inhibition in Xenopus embryos and can compensate for overexpression of exogenous BMP-4 (Cui et al. 1998). Finally, results from analyses of mice mutants null for specific furin-like convertases have strengthened the hypothesis that convertases process BMPs during embryonic development. Furin-deficient mice fail to show ventral closure, have defects in heart asymmetry, are defective in axial rotation, and die midway in development. TGFb-family members such as Nodal, Lefty-1, Lefty-2, and BMPs have been implicated in these processes, and mutants in downstream targets of BMPs phenocopy furin -/- mutants (Constam and Robertson, 2000a; Roebroek et al. 1998). Similarly, SPC4/PACE4 -/- mice demonstrate defects in left-right asymmetry as well, also presumably due to disruption of TGF-B signaling (Constam and Robertson, 2000b).

The role of furin-like protein convertases in processing TGFb like molecules was therefore deduced from analysis of the structure of the TGFb molecules and the overlapping expression patterns of convertases and BMPs in the mouse. It is therefore possible to similarly deduce a role for regulation of other developmentally significant proproteins by regulated processing. Although characterization of many molecules has revealed pro-proteins that may be proteolytically cleaved, other than BMP's in mammals and Vg1 in Xenopus, few studies have tested this hypothesis. Given that regulated proteolysis could represent a powerful mechanism for local and temporal control over protein activation, further studies are needed which specific address this possibility.

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