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Lawrence Shapiro

Publications and source records attributed to Lawrence Shapiro.

At least 19 recordsLinked to original sources
PubMed2006

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily.

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.

Amino Acid Sequence↗
PubMed2006

Type II cadherin ectodomain structures: implications for classical cadherin specificity.

Type I and II classical cadherins help to determine the adhesive specificities of animal cells. Crystal-structure determination of ectodomain regions from three type II cadherins reveals adhesive dimers formed by exchange of N-terminal beta strands between partner extracellular cadherin-1 (EC1) domains. These interfaces have two conserved tryptophan side chains that anchor each swapped strand, compared with one in type I cadherins, and include large hydrophobic regions unique to type II interfaces. The EC1 domains of type I and type II cadherins appear to encode cell adhesive specificity in vitro. Moreover, perturbation of motor neuron segregation with chimeric cadherins depends on EC1 domain identity, suggesting that this region, which includes the structurally defined adhesive interface, encodes type II cadherin functional specificity in vivo.

Amino Acid Sequence↗
PubMed2005

ADAM and Eph: how Ephrin-signaling cells become detached.

Ephrin ligands presented on one cell surface associate with their receptors on the surface of a juxtaposed cell, often resulting in cell-cell repulsion. In this issue of Cell, Janes et al. (2005) show that the ephrin ligand can be proteolytically released from its membrane tether by a complex on the opposing cell composed of the ephrin receptor and an ADAM metalloprotease.

ADAM Proteins↗
PubMed2005

Specificity of cell-cell adhesion by classical cadherins: Critical role for low-affinity dimerization through beta-strand swapping.

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell-cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by "swapping" the N-terminal beta-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that "closed" monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell-cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems.

Amino Acid Sequence↗
PubMed2005

Identification of a transiently exposed VE-cadherin epitope that allows for specific targeting of an antibody to the tumor neovasculature.

VE-cadherin is an adhesion molecule localized at the adherens junctions of endothelial cells. It is crucial for the proper assembly of vascular structures during angiogenesis and maintaining vascular integrity. We have studied 3 monoclonal antibodies (mAbs) against murine VE-cadherin that inhibit angiogenesis and tumor growth. Two of these, BV13 and 10G4, also disrupted normal vessels, resulting in severe vascular leakage, whereas the third, E4G10, did not. The goal of the current report was to identify the epitope of E4G10 and distinguish it from those of the disruptive mAbs. We mapped the epitope of E4G10 to within the first 10 amino acids of mature VE-cadherin and demonstrated that conserved tryptophan residues in this sequence are required for VE-cadherin-mediated trans-adhesion. The disruptive mAbs target a different epitope within amino acids 45 to 56, which structural homology modeling suggests is not involved in trans-adhesion. From our studies, we hypothesize that E4G10 can only bind the neovasculature, where VE-cadherin has not yet engaged in trans-adhesion and its epitope is fully exposed. Thus, E4G10 can inhibit junction formation and angiogenesis but is unable to target normal vasculature because its epitope is masked. In contrast, BV13 and 10G4 bind an epitope that is accessible regardless of VE-cadherin interactions, leading to the disruption of adherens junctions. Our findings establish the immediate N-terminal region of VE-cadherin as a novel target for inhibiting angiogenesis.

Angiogenesis Inhibitors↗
PubMed2005

New York-Structural GenomiX Research Consortium (NYSGXRC): a large scale center for the protein structure initiative.

Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.

Cloning, Molecular↗
PubMed2005

Crystal structures of the tryptophan repressor binding protein WrbA and complexes with flavin mononucleotide.

The tryptophan repressor binding protein WrbA binds to the tryptophan repressor protein TrpR. Although the biological role of WrbA remains unclear, it has been proposed to function in enhancing the stability of TrpR-DNA complexes. Sequence database analysis has identified WrbA as a founding member of a flavodoxin-like family of proteins. Here we present crystal structures of WrbA from Deinococcus radiodurans and Pseudomonas aeruginosa and their complexes with flavin mononucleotide. The protomer structure is similar to that of previously determined long-chain flavodoxins; however, each contains a conserved inserted region unique to the WrbA family. Interestingly, each WrbA protein forms a homotetramer with 222 symmetry, unique among flavodoxin-like proteins, in which each protomer binds one flavin mononucleotide cofactor molecule.

Amino Acid Sequence↗
PubMed2005

The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor.

Operons encoding stable toxins and their labile antidote are widespread in prokaryotes and play important roles in plasmid partitioning and cellular responses to stress. One such family of toxins MazF/ChpAK/PemK encodes an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific, but frequently occurring sites. Here we show that the Bacillus subtilis ydcE gene encodes a member of this family of RNases, which we have called EndoA. Overexpression of EndoA is toxic for bacterial cell growth and this toxicity is reversed by coexpression of the gene immediately upstream, ydcD. Furthermore, YdcD inhibits EndoA activity directly in vitro. EndoA has similar cleavage specificity to MazF and PemK and yields cleavage products with 3'-phosphate and 5'-hydroxyl groups, typical of EDTA-resistant degradative RNases. This is the first example of an antitoxin-toxin system in B. subtilis.

Bacillus subtilis↗
PubMed2004

Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.

Adipocytes hold the body's major energy reserve as triacylglycerols packaged in large lipid droplets. Perilipins, the most abundant proteins on these lipid droplets, play a critical role in facilitating both triacylglycerol storage and hydrolysis. The stimulation of lipolysis by beta-adrenergic agonists triggers rapid phosphorylation of perilipin and translocation of hormone-sensitive lipase to the surfaces of lipid droplets and more gradual fragmentation and dispersion of micro-lipid droplets. Because few lipid droplet-associated proteins have been identified in adipocytes, we isolated lipid droplets from basal and lipolytically stimulated 3T3-L1 adipocytes and identified the component proteins by mass spectrometry. Structural proteins identified in both preparations include perilipin, S3-12, vimentin, and TIP47; in contrast, adipophilin, caveolin-1, and tubulin selectively localized to droplets in lipolytically stimulated cells. Lipid metabolic enzymes identified in both preparations include hormone-sensitive lipase, lanosterol synthase, NAD(P)-dependent steroid dehydrogenase-like protein, acyl-CoA synthetase, long chain family member (ACSL) 1, and CGI-58. 17-beta-Hydroxysteroid dehydrogenase, type 7, was identified only in basal preparations, whereas ACSL3 and 4 and two short-chain reductase/dehydrogenases were identified on droplets from lipolytically stimulated cells. Additionally, both preparations contained FSP27, ribophorin I, EHD2, diaphorase I, and ancient ubiquitous protein. Basal preparations contained CGI-49, whereas lipid droplets from lipolytically stimulated cells contained several Rab GTPases and tumor protein D54. A close association of mitochondria with lipid droplets was suggested by the identification of pyruvate carboxylase, prohibitin, and a subunit of ATP synthase in the preparations. Thus, adipocyte lipid droplets contain specific structural proteins as well as lipid metabolic enzymes; the structural reorganization of lipid droplets in response to the hormonal stimulation of lipolysis is accompanied by increases in the relative mass of several proteins and the recruitment of additional proteins.

3T3-L1 Cells↗
PubMed2004

Structure of serine acetyltransferase from Haemophilus influenzae Rd.

The crystal structure of serine acetyltransferase (SAT) from Haemophilus influenzae Rd determined at 2.7 A resolution is presented. SAT is a member of a family of hexapeptide-containing transferases that contain six-residue tandem repeats (LIV)-G-X(4) that have been shown to form left-handed parallel beta-helices. In the current structure, each protomer is comprised of two domains: an N-terminal alpha-helical domain and a C-terminal left-handed parallel beta-helix domain. Although other members of this protein family are known to form trimeric structures, SAT forms a dimer of trimers in which the trimer interface is mediated through interactions between both the beta-helix domains and N-terminal domains; these trimers dimerize through contacts in the N-terminal domain. All dimer-of-trimer interactions are mediated through amino acids within an N-terminal extension common only to a subset of SATs, suggesting that members of this subfamily may also adopt hexameric structures. Putative active sites are formed by crevices between adjacent protomers in a trimer. Thus, six independent active sites exist in the hexameric enzyme complex.

Acetyltransferases↗
PubMed2004

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes.

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed2004

Disulfide-dependent multimeric assembly of resistin family hormones.

Resistin, founding member of the resistin-like molecule (RELM) hormone family, is secreted selectively from adipocytes and induces liver-specific antagonism of insulin action, thus providing a potential molecular link between obesity and diabetes. Crystal structures of resistin and RELMbeta reveal an unusual multimeric structure. Each protomer comprises a carboxy-terminal disulfide-rich beta-sandwich "head" domain and an amino-terminal alpha-helical "tail" segment. The alpha-helical segments associate to form three-stranded coiled coils, and surface-exposed interchain disulfide linkages mediate the formation of tail-to-tail hexamers. Analysis of serum samples shows that resistin circulates in two distinct assembly states, likely corresponding to hexamers and trimers. Infusion of a resistin mutant, lacking the intertrimer disulfide bonds, in pancreatic-insulin clamp studies reveals substantially more potent effects on hepatic insulin sensitivity than those observed with wild-type resistin. This result suggests that processing of the intertrimer disulfide bonds may reflect an obligatory step toward activation.

Adipocytes↗
PubMed2004

Structure of Escherichia coli YfdW, a type III CoA transferase.

Crystal structures are reported for free and coenzyme A (CoA) bound forms of the YfdW protein from Escherichia coli, a representative type III CoA transferase. The structures reveal a two-domain protomer with interdomain connections forming a ring-like structure with a large central hole. Two protomers associate to form a highly intertwined dimer in which the hole of each ring is filled by the partner molecule. Each protomer binds a single CoA molecule and these CoA-binding sites are distant from one another in the dimer.

Binding Sites↗
PubMed2004

Optimization of protein production in mammalian cells with a coexpressed fluorescent marker.

The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure for the direct selection of stable mammalian cell lines that express proteins of interest in high yield. Coexpression of a marker protein, such as green fluorescent protein, is linked to that of the desired protein through an internal ribosome entry site in the vector that is transfected into cells in culture. The coexpressed marker is used to select for highly expressing clonal cell lines. Applications are described to a membrane protein, the 5HT2c serotonin receptor, and to a secreted cysteine-rich protein, resistin. Besides providing an expeditious means for producing mammalian proteins for structural work, the resulting cell lines also readily support tests of functional properties and structure-inspired hypotheses.

Animals↗
PubMed2004

The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene.

Desmosomal cadherins are essential cell adhesion molecules present throughout the epidermis and other organs, whose major function is to provide mechanical integrity and stability to epithelial cells in a wide variety of tissues. We recently identified a novel desmoglein family member, Desmoglein 4 (Dsg4), using a positional cloning approach in two families with localized autosomal recessive hypotrichosis (LAH) and in the lanceolate hair (lah) mouse. In this study, we report cloning and identification of the rat Dsg4 gene, in which we discovered a missense mutation in a naturally occurring lanceolate hair (lah) rat mutant. Phenotypic analysis of lah/lah mutant rats revealed a striking hair shaft defect with the appearance of a lance head within defective hair shafts. The mutation disrupts a critical calcium binding site bridging the second and third extracellular domains of Dsg4, likely disrupting extracellular interactions of the protein.

Amino Acid Sequence↗
PubMed2004

Tubby proteins: the plot thickens.

The tubby mouse, which shows late-onset obesity and neurosensory deficits, arises from a mutation in the Tub gene. Tub shares homology with the genes for tubby-like proteins Tulp1, Tulp2 and Tulp3. Ablation of Tub, Tulp1 or Tulp3 causes disease phenotypes that are indicative of their importance in nervous-system function and development. Despite this importance, the biochemical functions of tubby-like proteins are only now beginning to be understood. At present, data indicate that tubby-like proteins might function as heterotrimeric-G-protein-responsive intracellular signalling factors, although an array of data also implicates them in other processes.

Adaptor Proteins, Signal Transducing↗