Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

The amyloid protein, A␤, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP). APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the A␤ domain (␤-cleavage), one within the A␤ domain (␣-cleavage), and one at the carboxyl terminus of the A␤ domain (␥-cleavage). The enzymes responsible for these activities have not been unambiguously identified. By the use of gene disruption (knockout), we now demonstrate that TACE (tumor necrosis factor ␣ converting enzyme), a member of the ADAM family (a disintegrin and metalloprotease-family) of proteases, plays a central role in regulated ␣-cleavage of APP. Our data suggest that TACE may be the ␣-secretase responsible for the majority of regulated ␣-cleavage in cultured cells. Furthermore, we show that inhibiting this enzyme affects both APP secretion and A␤ formation in cultured cells.The amyloid protein, A␤, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP) 1 (1-3). APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the A␤ domain (␤-cleavage), one within the A␤ domain (␣-cleavage), and one at the carboxyl terminus of the A␤ domain (␥-cleavage). The enzymes responsible for these activities have not been unambiguously identified.In most cells in culture, a fraction (10 -30%) of all APP undergoes ␣-cleavage (4 -7). This results in the secretion of the large extracellular domain of APP into the medium. This secreted APP (APP s ) is a major APP-related species found in cerebrospinal fluid and brain homogenates (8,9) and is thought to interact with components of the extracellular matrix and with receptors on cells. In cultured cells it has been shown that the fraction of APP that is converted to APP s can be increased by activating second messenger cascades including those involving protein kinase C, protein kinase A, mitogen-activated protein kinase, protein phosphatase 1, protein phosphatase 2B (calcineurin), and calcium (4 -6, 10 -14). In most cells in culture, activating protein kinase C causes the majority (80 -95%) of the APP to undergo ␣-cleavage ("regulated" ␣-cleavage). Stimulation of APP s formation and secretion by activating second messenger cascades is not due to the phosphorylation of APP (15, 16) but may be due to protein phosphorylation leading to alterations in the trafficking of APP (17) or in the activity of an ␣-secretase. Importantly, stimulating ␣-cleavage of APP leads to a significant decrease in A␤ formation (18 -20).The potential importance of regulated cleavage of APP is indicated by the ability of acetylcholine, a critical neurotransmitter altered in Alzheimer's disease, working through muscarinic receptors, to stimulate regulated cleavage (4,10,21,22). Activation of other metabotropic receptors also leads to activation of regulated secretion of APP (4,21,23,24). Regulated cleavage of APP appears to occur in vivo under conditions in which protein ...

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Voltage-gated sodium channels are concentrated in myelinated nerves at the nodes of Ranvier flanked by paranodal axoglial junctions. Establishment of these essential nodal and paranodal domains is determined by myelin-forming glia, but the mechanisms are not clear. Here, we show that two isoforms of Neurofascin, Nfasc155 in glia and Nfasc186 in neurons, are required for the assembly of these specialized domains. In Neurofascin-null mice, neither paranodal adhesion junctions nor nodal complexes are formed. Transgenic expression of Nfasc155 in the myelinating glia of Nfasc-/- nerves rescues the axoglial adhesion complex by recruiting the axonal proteins Caspr and Contactin to the paranodes. However, in the absence of Nfasc186, sodium channels remain diffusely distributed along the axon. Our study shows that the two major Neurofascins play essential roles in assembling the nodal and paranodal domains of myelinated axons; therefore, they are essential for the transition to saltatory conduction in developing vertebrate nerves.

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A method is described for searching protein sequence databases using tandem mass spectra of tryptic peptides. The approach uses a de novo sequencing algorithm to derive a short list of possible sequence candidates which serve as query sequences in a subsequent homology-based database search routine. The sequencing algorithm employs a graph theory approach similar to previously described sequencing programs. In addition, amino acid composition, peptide sequence tags and incomplete or ambiguous Edman sequence data can be used to aid in the sequence determinations. Although sequencing of peptides from tandem mass spectra is possible, one of the frequently encountered difficulties is that several alternative sequences can be deduced from one spectrum. Most of the alternative sequences, however, are sufficiently similar for a homology-based sequence database search to be possible. Unfortunately, the available protein sequence database search algorithms (e.g. Blast or FASTA) require a single unambiguous sequence as input. Here we describe how the publicly available FASTA computer program was modified in order to search protein databases more effectively in spite of the ambiguities intrinsic in de novo peptide sequencing algorithms.

Protein chemical cross-linking and mass spectrometry enable the analysis of protein-protein interactions and protein topologies, however complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein-protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms, and can identify cross-linked peptides using many different chemical cross-linkers, with or without heavy isotope labels. Kojak was used to analyze both novel and existing datasets, and was compared with existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms, and equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing datasets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods.