Il1

Ionizing radiation

IL-1 receptor

Poly-ubiquitin

Exterior Cytosol

Sequestered NF-kB

Sequestered NF-kB

Induces transcription of target genes

Induces transcription of target genes

Nucleus

Nucleus

Nuclear-localization signals

Free NF-kB 5

)/1 Proteasomal P65iP5ol degradation ^ J of I-kBœ

Nuclear-localization signals

I-kB protein bind active NF-kB in the nucleus and return it to the cytosol.

NF-kB stimulates transcription of more than 150 genes, including those encoding cytokines and chemokines that attract other immune-system cells and fibroblasts to sites of infection. It also promotes expression of receptor proteins that enable neutrophils (a type of white blood cell) to migrate from the blood into the underlying tissue (see Figure 6-30). In addition, NF-kB stimulates expression of iNOS, the inducible isoform of the enzyme that produces nitric oxide, which is toxic to bacterial cells, and of several anti-apoptotic proteins, which prevent cell death. Thus this single transcription factor coordinates and activates the body's defense either directly by responding to pathogens and stress or indirectly by responding to signaling molecules released from other infected or wounded tissues and cells.

Besides its roles in inflammation and immunity, NF-kB plays a key role during mammalian development. For instance, mouse embryos that cannot express one of the I-kB kinase subunits die at mid-gestation of liver degeneration caused by excessive apoptosis of cells that would normally survive; thus NF-kB is essential for normal development of this tissue. As we will see in Chapter 21, phosphorylation-dependent degradation of a cyclin kinase-dependent inhibitor plays a central role in regulating progression through the cell cycle in cerevisiae. It seems likely that phosphorylation-dependent protein degradation may emerge as a common regulatory mechanism in many different cellular processes.

Regulated Intramembrane Proteolysis Catalyzed by Presenilin 1 Activates Notch Receptor

Both Notch and its ligand Delta are transmembrane proteins with numerous EGF-like repeats in their extracellular domains. They participate in a highly conserved and important type of cell differentiation in both invertebrates and vertebrates, called lateral inhibition, in which adjacent and devel-opmentally equivalent cells assume completely different fates. This process, discussed in detail in Chapter 15, is particularly important in preventing too many nerve precursor cells forming from an undifferentiated layer of epithelial cells.

Notch protein is synthesized as a monomeric membrane protein in the endoplasmic reticulum, where it binds presenilin 1, a multispanning membrane protein; the complex travels first to the Golgi and then on to the plasma membrane. In the Golgi, Notch undergoes a proteolytic cleavage that generates an extracellular subunit and a transmembrane-cytosolic subunit; the two subunits remain noncovalently associated with each other in the absence of interaction with Delta residing on another cell. Binding of Notch to Delta triggers two proteolytic cleavages in the responding cell (Figure 14-29). The second cleavage, within the hydrophobic membrane-spanning region of Notch, is catalyzed by presenilin 1 and releases the Notch cytosolic segment, which immediately translocates to the nucleus. Such signal-induced regulated intramembrane proteolysis (RIP) also occurs in the response of cells to high cholesterol (Chapter 18) and to the presence of unfolded proteins in the endoplasmic reticulum (Chapter 16).

M FIGURE 14-29 Notch/Delta signaling pathway. The extracellular subunit of Notch on the responding cell is noncovalently associated with its transmembrane-cytosolic subunit. Binding of Notch to its ligand Delta on an adjacent signaling cell (step 1 ) first triggers cleavage of Notch by the membrane-bound metalloprotease TACE (tumor necrosis factor alpha converting enzyme), releasing the extracellular segment (step 2). Presenilin 1, an integral membrane protein, then catalyzes an intramembrane cleavage that releases the cytosolic segment of Notch (step 3). Following translocation to the nucleus, this Notch segment interacts with several transcription factors that act to affect expression of genes that in turn influence the determination of cell fate during development (step 4). [See M. S. Brown et al., 2000, Cell 100:391, and Y-M. Chan and Y Jan, 1999, Neuron 23:201.]

▲ FIGURE 14-30 Proteolytic cleavage of APP, a neuronal plasma membrane protein. (Left) Sequential proteolytic cleavage by a-secretase (step 1 ) and y-secretase (step 2) produces an innocuous membrane-embedded peptide of 26 amino acids. y-Secretase is a complex of several proteins, but the proteolytic site that catalyzes intramembrane cleavage probably resides within presenilin 1. (Right) Cleavage in the

In Drosophila the released intracellular segment of Notch forms a complex with a DNA-binding protein called Suppressor of Hairless, or Su(H), and stimulates transcription of many genes whose net effect is to influence the determination of cell fate during development. One of the proteins increased in this manner is Notch itself, and Delta production is correspondingly reduced (see Figure 15-38). As we see in Chapter 15, reciprocal regulation of the receptor and lig-and in this fashion is an essential feature of the interaction between initially equivalent cells that causes them to assume different cell fates.

Presenilin 1 (PS1) was first identified as the product of a gene that commonly is mutated in patients with an early-onset autosomal dominant form of Alzheimer's disease. A major pathologic change associated with Alzheimer's disease is accumulation in the brain of amyloid plaques containing aggregates of a small peptide containing 42 residues termed Ap42. This peptide is derived by proteolytic cleavage of APP (amyloid precursor protein), a cell-surface protein of unknown function expressed by neurons. APP actually undergoes cleavage by two pathways (Figure 14-30). In each pathway the initial cleavage occurs within the extracellular domain, catalyzed by a- or ^-secretase; y-secretase then catalyzes a second cleavage at the same intramembrane site in both pathways. The pathway initiated by a-secretase, which involves the same membrane-bound metalloprotease TACE that cleaves Notch, generates a 26-residue peptide that apparently does no harm. The pathway initiated by ^-secretase generates the pathologic Ap42. The missense mutations in presenilin 1 involved in Alzheimer's disease enhance the formation of the Ap42 peptide, leading to plaque formation and eventually to the death of neurons.

extracellular domain by p-secretase (step 1 ) followed by cleavage within the membrane by y-secretase generates the 42-residue Ap42 peptide that has been implicated in formation of amyloid plaques in Alzheimer's disease. In both pathways the cytosolic segment of APP is released into the cytosol, but its function is not known. [See W. Esler and M. Wolfe, 2001, Science 293:1449, and C. Haass and H. Steiner, 2002, Trends Cell Biol. 12:556.]

Evidence supporting the involvement of presenilin 1 in Notch signaling (see Figure 14-29) came from genetic studies in the roundworm C. elegans. Mutations in the worm homolog of presenilin 1 caused developmental defects similar to those caused by Notch mutations. Later work showed that mammalian Notch does not undergo signal-induced intramembrane proteolysis in mouse neuronal cells genetically missing presenilin 1. But whether presenilin 1 is the actual y-secretase protease or an essential cofactor of the "real" protease is not yet certain, since presenilin 1 is part of a large complex containing several other integral membrane proteins. Within its membrane-spanning segments, presenilin 1 has two aspartate residues in a configuration that resembles that of the two aspartates in the active site of water-soluble "aspartyl proteases," and mutation of either of these aspar-tate residues in presenilin 1 abolishes its ability to stimulate cleavage of Notch. Similarly, a battery of chemical protease inhibitors blocks cleavage of Notch and y-secretase cleavage of APP with the same potency, suggesting that the same protease is involved. Current data are thus consistent with the notion that presenilin 1 is the protease that cleaves both Notch and APP within their transmembrane segments. However, cleavage of both Notch and APP occurs at or near the plasma membrane, whereas the majority of presenilin is found in the endoplasmic reticulum. This finding suggests that presenilin may act in conjunction with other proteins in the unusual intramembrane proteolysis of Notch and APP. I

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