Gene name - nudel
Cytological map position - 65B1-65C1
Function - protease
Symbol - ndl
Genetic map position - 3-17
Classification - serine protease, WIID repeat, type A repeat
Cellular location - secreted, perivitelline fluid of the egg
nudel is one of over ten genes of so-called dorsal group of genes that constitute a signaling pathway responsible for dorsal-ventral polarity. Multiple signaling pathways, together called the dorsal group, establish dorsal-ventral polarity in the Drosophila embryo. The earlier acting of these pathways, involving the EGF receptor begins to function during oogenesis. The oocyte produces a dorsalizing signal to follicle cells, carried by the ligand Gürken, in order to define the polarity of the eggshell. After fertilization, a second signaling pathway, involving nudel and several other dorsal group genes and the gene cactus, generates a signal on the ventral side of the embryo. This signal is initiated by a protease cascade resulting in the ventral activation of the ligand Spätzle, which in turn activates the receptor Toll. This maternal pathway culminates in a graded ventral-to-dorsal distribution of the Dorsal protein, which controls the transcription activation or repression of a set of zygotic genes. A third signaling pathway defines patterning on the dorsal side of the embryo using a ligand encoded by decapentaplegic, a member of the TGFß family (Morisato, 1995 and references).
Nudel is one of three proteins produced by follicle cells that generate positional information leading to the activation of a protease cascade resulting in the ventral activation of Spätzle. Nudel is a mosaic protein; it consists of a complex domain structure, including a serine protease, suggesting that it is involved in multiple protein interactions while carrying out its role in the protease cascade. Two other proteins are produced by follicle cells: Pipe and Windbeutel. The functions of these proteins are unknown. The immediate targets of Nudel are Gastrulation defective, Snake and Easter, each known to be serine proteases. These three proteases act sequentially after Nudel in the protease cascade, with Easter being responsible for the proteolytic activation of Spätzle (Chasen, 1992).
From a knowledge of Nudel structure and biology, what can be learned about the role of Nudel in the proteolytic cascade? Nudel mRNA is found asymmetrically distributed in the follicle cells of a majority of unfertilized eggs. About a third of unfertilized eggs show a uniform distribution of Nudel mRNA; this suggests that the asymmetric distribution of Nudel is not the only cause of ventral activation of Spätzle. Nudel is a stable component of the perivitelline space. Experiments indicate that it is not freely diffusible; Nudel activity cannot be successfully transplanted in experiments that involve the transfer of perivitelline space fluid between embryos (Stein, 1991 and 1992). The fragile egg phenotype associated with most nudel mutations could mean that the nudel gene product is a structural component of the vitelline envelope. Nudel therefore has a role prior to fertilization in the successful completion of oogenesis.
Of particular interest is the question of how the protease function of Nudel becomes active. Like many other proteases, Nudel could be autoactivating, or Pipe could be responsible for its activation. pipe mutation results in a loss of the asymmetric Nudel mRNA distribution around the egg, suggesting an earlier involvement of Pipe in repressing elevation of Nudel mRNA in dorsal follicle cells (Hong, 1995).
The size and complexity of the Nudel protein suggests that it serves multiple functions: as anchor protein for proteases activated downstream in the cascade and as the trigger protein in the cascade. Nudel could act as the scaffold of a zymogen activation complex containing Gastrulation defective, Snake and Easter. Nudel would be an ideal scaffold protein, as it contains several domains implicated in protein-protein interactions (Hong, 1995).
The question remains: how does Nudel becomes activated, and how does Nudel function in a minority of eggs in which it is not asymmetrically distributed? For information on how dorsal and ventral follicle cells adopt different fates, resulting ultimately in the asymmetric distribution of Nudel, see Spätzle. For information on downstream events in the establishment of dorsal-ventral polarity, see Gurken.
A stretch of hydrophobic residues near the N-terminus of the protein could be part of a signal sequence seen in secreted and membrane-anchored proteins, since it is shortly followed by a possible signal sequence cleavage site. The Nudel protein is predicted to be extensively glycosylated. There are 23 potential sites for N-linked glycosylation throughout the molecule and two regions rich in serine and threonine that may be sites for O-linked glycosylation. Nudel also has three potential sites for glycosaminoglycan attachment, as seen in proteoglycans of the extracellular matrix (Hong, 1995).
Two types of sequence motifs are repeated in the protein. The first, consisting of nine residues and the sequence WI(I/L)D, is a novel motif called the WIID repeat. Nudel contains six copies of this repeat within the N-terminal threonine-rich region. The second repeat motif is a cysteine-rich sequence, consisting of about 40 residues, which was first identified in the ligand-binding domain of the low density lipoprotein (LDL) receptor. This motif, called a type A repeat, is found in a variety of proteins and is thought to mediate specific protein-protein interactions. The Nudel protein contains 11 copies of the type A repeat in four clusters.
The most striking features of the protein are two regions similar to the catalytic domains of extracellular serine proteases in the trypsin family. The more C-terminal protease domain region is similar only to the N-terminal half of the protease catalytic domain. This protease like domain lacks the residues of the catalytic triad (histidine, aspartic acid and serine) required for enzymatic activity, so it is not likely to be active as a protease.
In contrast, the region near the middle of Nudel is highly similar to the entire catalytic domain of a serine protease. In addition to the catalytic triad residues, there are eight cysteines that may form four intramolecular disulfide bonds within the catalytic pocket, as in chymotrypsin. Overall, the central protease domain of Nudel is most similar to the catalytic domains of vertebrate plasma kallikreins. Three key residues in the substrate-binding pocket of trypsin that determine amino acid specificity are conserved in the protease domain of Nudel, suggesting that the Nudel protease will cleave after lysine and arginine (Hong, 1995).
The protease domain of Nudel would presumably require proteolytic cleavage in order to become activated. Many serine proteases, such as those involved in blood clotting, are activated by cleavage at a precise site between an N-terminal regulatory region and the C-terminal catalytic domain. In Nudel, an arginine is just N-terminal to the conserved IVGG sequence at the beginning of the protease domain, implying that a proteinase specific for basic residues activates the protease activity of Nudel. Upon cleavage, the catalytic domain of Nudel is predicted to remain attached to the N-terminal region through a disulfide bond, as in the regulated proteases of blood clotting (Hong, 1995).
date revised: 5 MAR 97
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