HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone, to 2.1-Å resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

We have investigated the effect of type (3 transforming growth factor (TGF-() on the differentiation of skeletal muscle myoblasts. TGF-(3 potently (IDso 10 pM) prevents established cell lines and primary cultures of rat and chicken embryo myoblasts from fusing into multinucleated myotubes. Inhibition of morphological differentiation by TGF-,( correlates with inhibition of the expression of muscle-specific fiiRNAs and proteins, strong induction of extracellular matrix type I collagen and fibronectin, and a marked tendency of the treated myoblasts to aggregate into densely multilayered arrays or clusters. Myogenic differentiation can resume after removal of TGF-j3 from the medium. Examination of the time of action of TGF-(3 shows that myoblasts stochastically reach a point beyond which they become insensitive to the inhibitory action of TGF-(8. This resistance of committed myoblasts to the inhibitory action of TGF-.3 is not associated with any measurable change in the number or affinity of TGF-(3 receptors in those cells. The results indicate that TGF-(3 is a potent inhibitor of myogenesis and may regulate muscle development in vivo.Transforming growth factor ,3 (TGF-/3), a hormonally active polypeptide found in normal and transformed tissues, is a potent regulator of cell development (for review, see ref. 1). At picomolar concentrations TGF-P induces anchorageindependent growth of fibroblasts but inhibits the growth of certain tumor-derived as well as normal cells (2-6). In addition to its effects on cell proliferation, TGF-,B inhibits adipogenic differentiation without altering the growth rate of preadipocytes (7). Many cells, the growth or differentiation of which is regulated by TGF-,B, respond to this factor with a marked increase in the production and accumulation of the exetracellular matrix proteins fibronectin (8) and collagen (8,9). 'Available evidence suggests that the induction of an abundant extracellular matrix by TGF-,B mediates cellular responses to this factor such as the stimulation of anchorageindependent proliferation (8). These actions of TGF-P are presumably mediated by specific cell surface receptors: three structurally distinct cell surface glycoproteins that exhibit the properties of high-affinity receptors for TGF-P have been identified in mammalian and avian cells (10-12). It is not known whether all three receptor forms are involved in the mediation of TGF-,B actions or whether one receptor form is a signaling receptor, whereas the others have some other function(s).The widespread distribution of TGF-,3 and its receptors in different cell types and tissues suggests that this factor is involved in an ample spectrum of developmental processes in vivo. To obtain further information on the range of cellular targets for TGF-,B, we have investigated the effects of this factor on the differentiation of skeletal muscle myoblasts.These studies reveal a strong inhibitory action of TGF-,3 on myogenesis. METHODSL6Eq rat skeletal muscle myoblasts (13) were grown in Dulbecco's modified Eagle ...

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Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)–ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR–immunoglobulin G-Fcγ fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) may be important in triggering cell proliferation. PtdInsP2 also binds actin-binding proteins to regulate their activity, but it is not yet understood how this control is achieved. The protein alpha-actinin from striated muscle contains large amounts of endogenous PtdInsP2, whereas that from smooth muscle has only a little but will bind exogenously added PtdInsP2. In vitro alpha-actinin binds to F-actin and will crosslink actin filaments, increasing the viscosity of F-actin solutions. We report here that alpha-actinin from striated muscle is an endogenous PtdInsP2-bound protein and that the specific interaction between alpha-actinin and PtdInsP2 regulates the F-actin-gelating activity of alpha-actinin. Although the F-actin-gelating activity of alpha-actinin from smooth muscle is much reduced compared with that from striated muscle, exogenous PtdInsP2 can enhance the activity of smooth muscle alpha-actinin to the level seen in striated muscles. These results show that PtdInsP2 is present in striated muscle alpha-actinin and that it is necessary for alpha-actinin to realize its maximum gelating activity.

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We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP–induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract–induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP–depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP–dependent and –independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

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