Latent Transforming Growth Factor ��-binding Proteins and Fibulins Compete for Fibrillin-1 and Exhibit Exquisite Specificities in Binding Sites

Latent transforming growth factor (TGF) β-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFβ. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit “exquisite specificities,” a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.Fibrillin microfibrils are ubiquitous structural elements in the connective tissue. Fibrillin microfibrils provide organs with tissue-specific architectural frameworks designed to support the mature functional integrity of the particular organ. In addition, fibrillin microfibrils contribute to proper developmental patterning of organs by targeting growth factors to the right location in the extracellular matrix (1, 2).Molecules of fibrillin-1 (3), fibrillin-2 (4, 5), and fibrillin-3 (6) polymerize to form the backbone structure of microfibrils. Latent TGFβ-binding protein (LTBP)³-1 associates with fibrillin microfibrils in the perichondrium and in osteoblast cultures (7, 8), and LTBP-1 and LTBP-4 interact with fibrillin (9). Other proteins associated with fibrillin microfibrils include the fibulins (10, 11), microfibril-associated glycoprotein-1 and -2 (12, 13), decorin (14), biglycan (15), versican (16), and perlecan (17). It is likely that one function of these associated extracellular matrix molecules is to connect the fibrillin microfibril scaffold to other architectural elements in tissue- and organ-specific patterns.In addition to performing architectural functions, fibrillins bind directly to prodomains of bone morphogenetic proteins and growth and differentiation factors (18, 19) and LTBPs bring with them the small latent TGFβ complex (20), suggesting that the microfibril scaffold may position, concentrate, and control growth factor signaling. Studies of fibrillin-1 (Fbn1) and fibrillin-2 (Fbn2) mutant mice demonstrate that loss of fibrillins results in phenotypes associated with dysregulated TGFβ (21–23) or bone morphogenetic protein (24) signaling. Microfibril-associated glycoprotein-1 (Magp-1) null mice reveal phenotypes that may also be related to abnormal TGFβ signaling (25).In a previous study (9), we determined that the binding site for LTBP-1 and -4 is contained within a specific four-domain region of fibrillin-1. In this study, we performed additional experiments to more precisely define the LTBP binding site. At the same time, we compared binding of fibulins to fibrillin, because the region in fibrillin-1 that was suggested to contain the fibulin binding site (11) was very close to our region of interest for LTBP binding. Our results demonstrate that LTBPs and fibulins compete for binding to fibrillin-1. However, the proteins tested (LTBP-1, LTBP-4, fibulin-2, fibulin-4, and fibulin-5) displayed “exquisite specificities” in their interactions with fibrillin-1.To test the potential significance of these interactions with fibrillin-1, we investigated matrix incorporation of LTBPs in cell cultures obtained from wild type, Fbn1 null, Fbn2 null, fibulin-2 (Fbln-2) null, and fibulin-4 (Fbln-4) null mice. In addition, we examined the distribution of LTBPs in Fbn1 null and Fbn2 null mice.     

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin ��V��3

Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin α_Vβ₃ antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin α_Vβ₃ as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion.Cellular interactions with extracellular matrix proteins are vital for cell survival and tissue maintenance. The attachment of cells to their extracellular matrix (ECM)³ is often mediated by cell surface integrins. As such, integrins are involved in many biological functions such cell migration and proliferation, tissue organization, wound repair, development, and host immune responses. In addition to roles under normal physiological conditions, integrins are involved in the pathogenesis of diseases such as arthritis, cardiovascular disease, inflammation, microbial and parasitic infection, and cancer. Integrins are a family of heterodimeric transmembrane receptors containing one α subunit and one β subunit (1). Often integrins bind to ECM proteins via short RGD motifs within the matrix protein (2). In addition to an RGD motif, fibronectin also contains an upstream PHSRN synergy sequence, which is required for full integrin binding activity (3).Elastin confers elasticity on all vertebrate elastic tissues including arteries, lung, skin, vocal fold, and elastic cartilage (4). Elastin comprises ∼90% of the elastic fiber and is intermingled with fibrillin-rich microfibrils (5). There is a single human tropoelastin gene in which alternative splicing can result in the loss of domains 22, 23, 24, 26A, 30, 32, and 33 (4). Elastin is made from the secreted monomer tropoelastin, which is a 60–72-kDa protein containing repeating hydrophobic and cross-linking domains. Hydrophobic domains are rich in GVGVP, GGVP, and GVGVAP repeats, which can associate by coacervation (6). This association results in structural changes and increased α-helical content (7). The cross-linking domains are lysine-rich. Occasionally these residues are modified to allysine through the activity of members of the family of lysyl oxidase (LOX) and four LOX-like enzymes. During coacervation the allysine and other allysines or specific lysine side chains come into close proximity, allowing nonenzymatic condensation reactions to occur, forming desmosine or isodesmosine cross-links (4). This process gives a highly stable cross-linked elastin matrix which has a half-life of ∼70 years. Members of the serine, aspartate, cysteine, and matrix metalloproteinase families of proteases can degrade elastin (8). The resulting elastin peptides have effects on ECM synthesis and cell attachment, migration, and proliferation (9).The consequences of mutated or hemizygous elastin in the hereditary, connective tissue disorders cutis laxa, supravalvular aortic stenosis, and Williams-Beuren syndrome highlight the elastins essential role in elastic tissue function (10). Elastin is the major protein in large elastic blood vessels such as the aorta, where it is likely to inhibit the proliferation of vascular smooth muscle cells and so preventing vessel occlusion (11), which is a major cause of death in developed countries. Previous studies have shown that human and bovine tropoelastin can bind directly to a variety of cell types directly through a number of cell surface receptors (12–14) and also bind indirectly to cells through ECM proteins such as fibulin-5 (15, 16).A mechanism by which elastin binds to cells is via the 67-kDa elastin-binding protein (EBP), which is a peripheral membrane splice variant of β-galactosidase. The EBP forms a complex with the integral membrane proteins carboxypeptidase A and sialidase, forming a transmembrane elastin receptor (12). The binding site for the EBP has been mapped to the consensus sequence XGXXPG within elastin and in particular to VGVAPG within exon 24 (17). The binding of elastin to the EBP results in cell morphological changes (18, 19), chemotaxis (20), decreased cell proliferation (21), and angiogenesis (22). Knockouts of β-galactosidase, which remove the EBP, display correctly deposited elastin (27). Additionally tropoelastin actively promotes cell adhesion, whereas VGVAPG does not. These observations imply that receptors other than EBP can interact with elastin.Other studies have proposed a second mechanism involving the necessity of cell surface heparan and chondroitin sulfate-containing glycosaminoglycans for bovine chondrocyte interaction with bovine tropoelastin (14). Peptide binding analysis implicated the last 17 amino acids at the C terminus of bovine tropoelastin in this cell adhesive activity, with higher binding requiring the C-terminal 25 amino acids. This region is of interest, as in humans a mutation of Gly-773 to Asp in exon 33 results in blocked elastin network assembly and modulates cell binding to a peptide corresponding to exons 33 and 36 of human tropoelastin (28). Indeed Broekelmann et al. (14) have shown that synthetic peptides containing the C-terminal 29 amino acids of bovine tropoelastin possess cell adhesive activity; however, when the G773D mutation was incorporated into the peptide, it prevented cell adhesion to that peptide.Although tropoelastin does not contain an RGD motif, other data identified a third mechanism involving direct interaction between integrin α_vβ₃ and human tropoelastin (13, 29). This interaction was also localized to the C-terminal domains of tropoelastin.More recent data has shown that human umbilical vein endothelial cells can adhere to recombinant fragments of human tropoelastin (30, 31). In contrast to other data, regions encoded by the N-terminal exons (1–18), the central exons (18–27), and the C-terminal exons (18–36) all supported human umbilical vein endothelial cell attachment.Although a previous study has shown a direct interaction between purified integrin α_vβ₃ and human tropoelastin (13), the integrin dependence of cell adhesion to tropoelastin had not been demonstrated. Here we demonstrate that human dermal fibroblasts adhere to recombinant human tropoelastin and that inhibitors of the elastin-binding protein and cell surface heparan sulfate have no effect on cell adhesion. In contrast, cell adhesion was dependent upon the presence of divalent cations, indicating integrin dependence. Inhibitory monoclonal antibodies identified integrin α_Vβ₃ as the major receptor necessary for fibroblast adherence and spreading onto human tropoelastin. The binding motif for integrin-mediated cell adhesion is unknown; therefore, through the use of synthetic peptides, the adhesive activity was localized to the extreme C-terminal GRKRK motif of tropoelastin. This data present a novel mechanism for cell adhesion to human tropoelastin and identify a novel integrin binding motif within tropoelastin.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Defining Elastic Fiber Interactions by Molecular Fishing: AN AFFINITY PURIFICATION AND MASS SPECTROMETRY APPROACH*

Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix.Mass spectrometry is emerging as a powerful approach to identify protein interaction partners in molecular complexes. We have developed an affinity purification and mass spectrometry strategy that is applicable to the analysis of molecular interactions of extracellular matrix complexes. The extracellular matrix provides structural support to tissues and profoundly influences cell survival, proliferation, migration, and phenotypic state. It is a complex multimolecular and three-dimensional milieu that comprises assembled networks of tissue-specific combinations of structural and cell-adhesive glycoproteins, proteoglycans, and cross-linking enzymes. The matrix also sequesters numerous growth factors and cytokines, thereby controlling their bioavailability. Delineating the molecular nature of the fundamental interacting networks within complex extracellular matrices is a challenging task. Here, mass spectrometry has given new insights into elastic fiber interactions.Elastic fibers are essential structural elements of the extracellular matrix of dynamic connective tissues such as blood vessels, lungs, skin, and ligaments, endowing these tissues with elastic recoil (1, 2). Their importance is emphasized by elastic fiber defects that cause severe acquired diseases such as aortic aneurysms and pulmonary emphysema and life-threatening heritable disorders such as Marfan syndrome, supravalvular stenosis, and cutis laxa. These fibers are extensive multimolecular assemblies that adopt intricate tissue-specific architectural arrangements. At the morphological level, the fibers comprise a cross-linked elastin core and an outer mantle of fibrillin microfibrils. It has proved challenging to define the composition of tissue elastic fibers biochemically. Cross-linked elastin is highly insoluble and its isolation from tissues requires extreme conditions of hot alkali, which destroys other proteins (2). The efficient extraction of tissue microfibrils requires collagenase and other proteolytic activities that may destroy associated molecules (3). Despite these difficulties, a number of associated proteins, including MAGP-1,1 βigH3, fibulins, and lysyl oxidases (LOX and LOXL (also known as LOXL1)), as well as latent TGFβ-binding proteins (LTBPs), collagen VIII, and emilin-1 have been identified in biochemical and/or colocalization studies (1).Fibrillins are very large glycoproteins (350 kDa) containing 43 calcium-binding epidermal growth factor-like domains and seven TGFβ-binding protein-like (8-cysteine) domains (4). Fibrillin-1 is the more abundant isoform; fibrillin-2 is mainly expressed during development (5, 6). Tropoelastin, the secreted soluble form of elastin, comprises alternating hydrophobic and lysine-rich cross-linking domains. LOX and LOXL are copper-dependent amine oxidases that cross-link elastin through the oxidative deamination of specific lysines (7–9). Elastin is mainly expressed and deposited early in life and undergoes very little turnover in healthy tissues (2). MAGP-1 is a microfibril-associated glycoprotein that binds fibrillin-1 and elastin (10, 11) but is not essential for elastic fiber formation (12). βigH3 was originally identified as a matrix protein, MP78/70, in tissue extracts that solubilized elastin-associated microfibrils (13, 14). Fibulin-4 and -5 play essential roles in elastic fiber formation (15, 16), most likely by regulating elastin deposition onto microfibrils (17, 18). Fibulin-2 interacts with fibrillin-1 (19) but is not essential for elastic fiber formation (20). Fibulin-1-null mice, among other symptoms, display anomalies of aortic arch arteries and hemorrhagic blood vessels, suggesting some involvement in elastic fiber biology (21). Fibulin-3 (also known as Efemp1)-deficient mice exhibit early aging and herniation associated with reduced elastic fiber integrity (22). Collagen VIII and emilin-1 also colocalize to elastic fibers (23, 24).The assembly of microfibrils and elastic fibers remains incompletely understood. We and others recently showed that assembly of the microfibril component is orchestrated by the cell surface through interactions with fibronectin and integrin receptors (25, 26). Heparan sulfate, an abundant pericellular glycosaminoglycan chain attached to syndecan and glypican proteoglycan receptors, also critically influences microfibril formation (27–29). Elastin deposition and stabilization on microfibrils require fibulins and the cross-linking enzymes LOX and/or LOXL.To obtain new insights into the molecular interactions of elastic fibers and how they are integrated into their surrounding matrix, we conducted a detailed affinity capture LC-MS/MS analysis of molecules that interact in culture specifically with four His₆-tagged recombinant human elastic fiber molecules (fibrillin-1, MAGP-1, fibulin-5, and LOX). Tropoelastin was not used as bait because of its highly adhesive nature. Our protocol proved to be an effective strategy for defining specific interactions of elastic fiber molecules in the extracellular matrix. Efficacy was demonstrated through confirmation of known interactions and validation of novel extracellular matrix protein-protein interactions. This approach further allowed us to predict secondary elastic fiber interactions, giving powerful insights into the molecular networks that sustain elastic fibers within higher order extracellular matrices.     

Fibulin 5 Forms a Compact Dimer in Physiological Solutions

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca²⁺ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.Fibulins are a family of seven extracellular matrix glycoproteins, some of which associate with elastic fibers and basement membranes (1, 2). They are involved in the assembly, organization, and stabilization of macromolecular complexes (3). Fibulins contain arrays of cbEGF²-like domains and a fibulin-type C-terminal (Fc) module (4). Fibulins 3–5 have a modified N-terminal cbEGF domain, followed by five cbEGF domains (4).Fibulin 5 (supplemental Fig. S1) is highly expressed in developing arteries with a low expression in adult vessels that is up-regulated following vascular injury and in atherosclerosis (5, 6). Expression has been detected in other elastin-rich tissues, including aorta, skin, uterus, lung, heart, ovary, and colon (5, 6). The extensibility of such tissues is provided by elastic fibers (7), and aging is associated with a loss of elasticity (8). Fibulin 5 is essential for elastinogenesis. The fibulin 5 knock-out mouse exhibits disorganized elastic fibers resulting in severe elastinopathies, with loose skin, vascular abnormalities, and emphysematous lungs. Similar changes are seen in an aged phenotype (9, 10). Mutations in fibulin 5 lead to the elastin disorder cutis laxa (11–13) and have been associated with age-related macular degeneration (14, 15).It has been shown that fibulin 5 binds elastic fibers (16) and interacts with tropoelastin (10), fibrillin 1 (17), lysyl oxidase-like protein 1 (18), -2, and -4 (19), latent transforming growth factor-β-binding protein 2 (19), emilin 1 (20), apolipoprotein (a) (21), and superoxide dismutase (22). Through an RGD motif fibulin 5 interacts with integrins (6, 9, 23).The assembly of elastic fibers is a complex hierarchical process. A model proposes that fibulin 5 associates with microfibrils via interactions with fibrillin 1; tropoelastin molecules bind fibulin 5 and coacervate, and lysyl oxidase-like protein 1 enzymes cross-link tropoelastin to form mature elastin (7, 16). Data that support this model indicate that fibulin 5 potentially increases the coacervation of tropoelastin, enhancing elastic fiber formation (24). However, other data suggest that fibulin 5 slows the maturation of elastin assemblies (25).Rotary-shadowing EM has suggested that fibulin 5 exists as a short rod with a globular domain at one end (26). We used size-exclusion column multiangle laser light scattering (SEC-MALLS), small angle x-ray scattering (SAXS), EM single particle analysis, analytical ultracentrifugation (AUC), CD, and isoelectric focusing to investigate the structures of fibulin 5 in monomeric and dimeric form, and the equilibrium between the two forms.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Evidence that Antibody-Mediated Neutralization of Human Immunodeficiency Virus Type 1 by Sera from Infected Individuals Is Independent of Coreceptor Usage

Human immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1α, and MIP-1β in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous Δ32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS, utilizes the HLA class II receptor, CD4, as its primary receptor to gain entry into cells (17, 30). Entry is initiated by a high-affinity interaction between CD4 and the surface gp120 of the virus (32). Subsequent to this interaction, conformational changes that permit fusion of the viral membrane with cellular membranes occur within the viral transmembrane gp41 (9, 58, 59). In addition to CD4, one or more recently described viral coreceptors are needed for fusion to take place. These coreceptors belong to a family of seven-transmembrane G-protein-coupled proteins and include the CXC chemokine receptor CXCR4 (3, 4, 24, 44), the CC chemokine receptors CCR5 (1, 12, 13, 18, 21, 23, 45) and, less commonly, CCR3 and CCR2b (12, 21), and two related orphan receptors termed BONZO/STRL33 and BOB (19, 34). Coreceptor usage by HIV-1 can be blocked by naturally occurring ligands, including SDF-1 for CXCR4 (4, 44), RANTES, MIP-1α, and MIP-1β in the case of CCR5 (13, 45), and eotaxin for CCR3 (12).The selective cellular tropisms of different strains of HIV-1 may be determined in part by coreceptor usage. For example, all culturable HIV-1 variants replicate initially in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), but only a minor fraction are able to infect established CD4⁺ T-cell lines (43). This differential tropism is explained by the expression of CXCR4 together with CCR5 and other CC chemokine coreceptors on PBMC and the lack of expression of CCR5 on most T-cell lines (5, 10, 19, 35, 39, 50, 53). Indeed, low-passage field strains (i.e., primary isolates) of HIV-1 that fail to replicate in T-cell lines use CCR5 as their major coreceptor and are unable to use CXCR4 (1, 12, 18, 21, 23, 28). Because these isolates rarely produce syncytia in PBMC and fail to infect MT-2 cells, they are often classified as having a non-syncytium-inducing (NSI) phenotype. Primary isolates with a syncytium-inducing (SI) phenotype are able to use CXCR4 alone or, more usually, in addition to CCR5 (16, 20, 51). HIV-1 variants that have been passaged multiple times in CD4⁺ T-cell lines, and therefore considered to be laboratory adapted, exhibit a pattern of coreceptor usage that resembles that of SI primary isolates. Most studies have shown that the laboratory-adapted strain IIIB uses CXCR4 alone (3, 13, 20, 24, 51) and that MN and SF-2 use CXCR4 primarily and CCR5 to a lesser degree (11, 13). Sequences within the V3 loop of gp120 have been shown to be important, either directly or indirectly, for the interaction of HIV-1 with both CXCR4 (52) and CCR5 (12, 14, 54, 60). This region of gp120 contains multiple determinants of cellular tropism (43) and is a major target for neutralizing antibodies to laboratory-adapted HIV-1 but not to primary isolates (29, 46, 57).It has been known for some time that the ability of sera from HIV-1-infected individuals to neutralize laboratory-adapted strains of HIV-1 does not predict their ability to neutralize primary isolates in vitro (7). In general, the former viruses are highly sensitive to neutralization whereas the latter viruses are neutralized poorly by antibodies induced in response to HIV-1 infection (7, 43). Importantly, neutralizing antibodies generated by candidate HIV-1 subunit vaccines have been highly specific for laboratory-adapted viruses (26, 37, 38). In principle, the dichotomy in neutralization sensitivity between these two categories of virus could be related to coreceptor usage. To test this, we investigated whether the use of CXCR4 in the absence of CCR5 would render SI primary isolates highly sensitive to neutralization in vitro by sera from HIV-1-infected individuals. Two similar studies using human monoclonal antibodies and soluble CD4 have been reported (31a, 55).     

Question Processing and Clustering in INDOC: A Biomedical Question Answering System

The exponential growth in the volume of publications in the biomedical domain has made it impossible for an individual to keep pace with the advances. Even though evidence-based medicine has gained wide acceptance, the physicians are unable to access the relevant information in the required time, leaving most of the questions unanswered. This accentuates the need for fast and accurate biomedical question answering systems. In this paper we introduce INDOC—a biomedical question answering system based on novel ideas of indexing and extracting the answer to the questions posed. INDOC displays the results in clusters to help the user arrive the most relevant set of documents quickly. Evaluation was done against the standard OHSUMED test collection. Our system achieves high accuracy and minimizes user effort.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]