ADAMs (a disintegrin and metalloproteinase) are sheddases possessing extracellular metalloproteinase/disintegrin/cysteine-rich (MDC) domains. ADAMs uniquely display both proteolytic and adhesive activities on the cell surface, however, most of their physiological targets and adhesion mechanisms remain unclear. Here for the first time, we reveal the ADAMs' MDC architecture and a potential target-binding site by solving crystal structures of VAP1, a snake venom homolog of mammalian ADAMs. The D-domain protrudes from the M-domain opposing the catalytic site and constituting a C-shaped arm with cores of Ca 2 þ ions. The disintegrin-loop, supposed to interact with integrins, is packed by the C-domain and inaccessible for protein binding. Instead, the hyper-variable region (HVR) in the C-domain, which has a novel fold stabilized by the strictly conserved disulfide bridges, constitutes a potential protein-protein adhesive interface. The HVR is located at the distal end of the arm and faces toward the catalytic site. The C-shaped structure implies interplay between the ADAMs' proteolytic and adhesive domains and suggests a molecular mechanism for ADAMs' target recognition for shedding.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

Plant B-type cyclin genes are expressed specifically in late G2- and M-phases during the cell cycle. Their promoters contain a common cis-acting element, called the MSA (M-specific activator) element, that is necessary and sufficient for periodic promoter activation. This motif also is present in the tobacco kinesin-like protein gene NACK1, which is expressed with timing similar to that of B-type cyclin genes. In this study, we show that G2/M-phase-specific activation of the NACK1 promoter also is regulated by the MSA element, suggesting that a defined set of G2/M-phase-specific genes are coregulated by an MSA-mediated mechanism. In a search for MSA binding factors by yeast one-hybrid screening, we identified three different Myb-like proteins that interact specifically with the MSA sequence. Unlike the majority of plant Myb-like proteins, these Myb proteins, NtmybA1, NtmybA2, and NtmybB, have three imperfect repeats in the DNA binding domain, as in animal c-Myb proteins. During the cell cycle, the level of NtmybB mRNA did not change significantly, whereas the levels of NtmybA1 and A2 mRNAs fluctuated and peaked at M-phase, when B-type cyclin genes were maximally induced. In transient expression assays, NtmybA1 and A2 activated the MSA-containing promoters, whereas NtmybB repressed them. Furthermore, expression of NtmybB repressed the transcriptional activation mediated by NtmybA2. Our data show that a group of plant Myb proteins that are structurally similar to animal c-Myb proteins have unexpected roles in G2/M-phase by modulating the expression of B-type cyclin genes and may regulate a suite of coexpressed genes.

Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/ Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, ϳ50%) as well as in inhibition of sorbitol-induced glucose transport (by ϳ35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4.Insulin stimulates glucose transport into muscle and adipose tissue by inducing the translocation of vesicles containing the glucose transporter GLUT4 from the intracellular compartment to the plasma membrane (1, 2). This process is thought to be a major contributor to the mechanism by which insulin reduces the blood concentration of glucose. The binding of insulin to its receptor on the surface of target cells results in receptor autophosphorylation and receptor-mediated tyrosine phosphorylation of several additional proteins, including insulin receptor substrates 1-4 (IRS1 to IRS4).1 The phosphorylated IRS proteins then bind other proteins, such as phosphoinositide (PI) 3-kinase, SHP-2, and GRB2, that contain SRC homology 2 (SH2) domains (3). PI 3-kinase is thought to play a role in the insulin-induced translocation of GLUT4 (4, 5); however, the mechanism by which activation of PI 3-kinase results in GLUT4 translocation remains unclear (6).The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) hypothesis was initially proposed to explain the process of neurotransmitter secretion (7,8). According to this hypothesis, the docking and fusion of synaptic vesicles at the plasma membrane are initiated by the interaction of proteins, known as v-SNAREs (synaptobrevin/ VAMP), located on the vesicle surface with corresponding proteins, known as t-SNAREs (syntaxin, SNAP-25), located on the target membrane. Membrane fusion is subs...

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Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-⌬C8 (amino acids 1 to 202) and SNAP23-⌬C49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-⌬C49 bound to neither syntaxin4 nor VAMP2, the SNAP23-⌬C8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-⌬C8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-⌬C8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-⌬C49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).A primary function of insulin is to stimulate the transport of glucose into target tissues, prominent among which are skeletal muscle, cardiac muscle, and adipose tissue. Insulin achieves this effect by inducing the translocation of GLUT4 glucose transporters from an intracellular vesicular compartment to the plasma membrane. Under basal conditions, GLUT4 cycles slowly between this intracellular compartment and the plasma membrane (1, 2). However, activation of insulin receptors triggers a large increase in the rate of exocytosis of GLUT4-containing vesicles and a smaller decrease in the rate of GLUT4 internalization by endocytosis (3-5), with the former action likely contributing most to the insulin-induced increase in the amount of GLUT4 in the plasma membrane (6).Intracellular membrane fusion is mediated by evolutionarily conserved membrane proteins known as soluble N-ethylmaleimide-sensitive factor (NSF) 1 attachment protein receptors (SNAREs) (7,8). SNARE proteins that contribute to neuronal exocytosis include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the plasma membrane proteins synaptosome-associated 25-kDa protein and syntaxin1A. These proteins readily assemble into a stable ternary complex; however, disassembly of this complex can be reversibly induced by the ATPase NSF in conjunction with soluble cofactors termed SNAPs (soluble NSF-att...

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A double-blind, multi-center study was performed on patients with HTLV-I-associated myelopathy (HAM) to evaluate the therapeutic effect of treatment with natural interferon-alpha (HLBI). Forty-eight HAM patients were enrolled and treated with either 0.3 MU (n = 15), 1.0 MU (n = 17), or 3.0 MU (n = 16) of HLBI for 28 days. Clinical evaluation included motor dysfunction, urinary disturbances, and changes of neurologic signs. The frequency of therapeutic response judged as excellent to good 4 weeks after starting therapy and 4 weeks after completion of therapy were 7.1% (1 of 14) and 8.3% (1 of 12) in the 0.3-MU group, 23.5% (4 of 17) and 26.7% (4 of 15) for the 1.0-MU group, and 66.7% (10 of 15) and 61.5% (8 of 13) for the 3.0-MU group. The therapeutic benefit in the 3.0-MU group was significantly higher than in the 0.3-MU group. There was no significant difference in the incidence of symptomatic side effects between groups. Abnormal laboratory data were obtained for some patients in the 1.0-MU and 3.0-MU groups; however, the treatment schedule could be continued in most patients. These results suggest that HAM patients may be safely treated with HLBI 3.0 MU every day for 4 weeks with favorable clinical effects.