Tumour necrosis factor (tumour necrosis factor-alpha/cachectin) plays a critical role in certain physiological defensive responses but causes severe damage to the host organism when produced in excess. There are two forms of tumour necrosis factor, a type II membrane protein of relative molecular mass 26,000 (26K) and a soluble, 17K form generated from the cell-bound protein by proteolytic cleavage. The two forms of tumour necrosis factor and lymphotoxin-alpha (tumour necrosis factor-beta/lymphotoxin), a related protein, have similar but apparently not identical biological activities. A therapeutic agent which inhibited the release of tumour necrosis factor, but did not reduce the cell-associated activity or the level of lymphotoxin-alpha, might preserve the benefits of these cytokines while preventing tumour necrosis factor-induced damage. Here we describe a potent inhibitor of tumour necrosis factor processing and report that it protects mice from a lethal dose of endotoxin.

Tumor necrosis factor-␣ (TNF␣) is a cytokine that induces protective inf lammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNF␣ is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNF␣-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 Å crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin͞ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNF␣ cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNF␣ cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3 pocket merging with the S1 specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNF␣-induced pathophysiological effects, and might also help to control related shedding processes.Tumor necrosis factor-␣ (TNF␣) (1), a major immunomodulatory and proinflammatory cytokine, is synthesized as a 223-aa membrane-anchored precursor. The soluble form of TNF␣, comprising the C-terminal two-thirds of this precursor, is released into extracellular space by limited proteolysis at the Ala-76 3 Val-77 bond. The proteinase responsible for this cleavage, called TACE or ADAM 17, has recently been identified (2, 3) as a zinc-endopeptidase consisting of a multidomain extracellular part, an apparent transmembrane helix and an intracellular C-terminal tail. The extracellular part comprises an N-terminal pro domain, a 259-residue catalytic domain, and a Cys-rich moiety that has been hypothesized to be composed of a disintegrin-like, an epidermal growth factorlike, and a crambin-like domain (2). Its polypeptide sequence, in particular, that accounting for the catalytic domain, indicates some similarity with other metzincins (4, 5), especially with the adamalysins͞ADAMs (6-8) (a protein family comprising snake venom metalloproteinases and membraneanchored surface proteins containing an adamalysin-like catalytic domain) and the matrix metalloproteinases (MMPs). In comparison to enzymes in these families, however, the polypeptide chain of the TACE catalytic domain is clearly longer and is stable in the absence of calcium. Further, in contrast to the MMPs, TACE is relatively insensitive to the tissue inhibitor of metalloproteinases-1 (TIMP-1) (9) and exhibits a different inhibition pattern toward synthetic inhibitors (9-12). In contrast to the MMPs, TACE cleaves a 12-mer peptide spanning the cleavage site in...

The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor indicated that it is a novel member of the plexin family and identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on, and induced cytokine production from, human monocytes. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs.

The chemisorption of methane dissociation intermediates, CH x (x = 0−3), on Pt{110}(1 × 2) has been investigated using calculations based on density functional theory. For all species considered, the most stable adsorption site identified on the missing-row reconstructed (1 × 2) surface is the site that not only completes the tetravalency of the carbon atom but also involves the maximum number of ridge Pt atoms for a site of that type. Thus, methyl (CH3) preferentially occupies the ridge atop site; methylene (CH2), the ridge bridge site; methylidyne (CH), the fcc 3-fold site on the {111} microfacet; and carbon, the pseudosubsurface 4-fold site at the bottom of the trough. A comparison of the relative stability of the chemisorbed CH x (x = 0−3) species reveals that CH is the most stable dissociation intermediate on Pt{110}(1 × 2).

Nuclear intermediate filament proteins, called lamins, form a meshwork that lines the inner surface of the nuclear envelope. Lamins contain three domains: an N-terminal head, a central rod and a C-terminal tail domain possessing an Ig-fold structural motif. Lamins are classified as either A- or B-type based on structure and expression pattern. The Drosophila genome possesses two genes encoding lamins, Lamin C and lamin Dm0, which have been designated A- and B-type, respectively, based on their expression profile and structural features. In humans, mutations in the gene encoding A-type lamins are associated with a spectrum of predominantly tissue-specific diseases known as laminopathies. Linking the disease phenotypes to cellular functions of lamins has been a major challenge. Drosophila is being used as a model system to identify the roles of lamins in development. Towards this end, we performed a comparative study of Drosophila and human A-type lamins. Analysis of transgenic flies showed that human lamins localize predictably within the Drosophila nucleus. Consistent with this finding, yeast two-hybrid data demonstrated conservation of partner-protein interactions. Drosophila lacking A-type lamin show nuclear envelope defects similar to those observed with human laminopathies. Expression of mutant forms of the A-type Drosophila lamin modeled after human disease-causing amino acid substitutions revealed an essential role for the N-terminal head and the Ig-fold in larval muscle tissue. This tissue-restricted sensitivity suggests a conserved role for lamins in muscle biology. In conclusion, we show that (1) localization of A-type lamins and protein-partner interactions are conserved between Drosophila and humans, (2) loss of the Drosophila A-type lamin causes nuclear defects and (3) muscle tissue is sensitive to the expression of mutant forms of A-type lamin modeled after those causing disease in humans. These studies provide new insights on the role of lamins in nuclear biology and support Drosophila as a model for studies of human laminopathies involving muscle dysfunction.