Heterogeneity of β2-Microglobulin in Human Urine

Purification and characterization of β₂-microglobulin from human urine was performed. The yield was 30.1%, and 150.4 mg of β₂-microglobulin was obtained. The final preparation of β₂-microglobulin obtained showed three bands on disc gel electrophoresis at pH 9.5, and all of them have immunological activity. However, these three bands migrated as a single band on disc gel electrophoresis at pH 4.3. It is concluded that the three bands observed on disc gel electrophoresis at pH 9.5 were charge isomers. The isoelectric points of isomers were determined by isotachophoresis and two of them were 5.4 and 5.9 respectively, while the other one was not determined.     

Human α2macroglobulin

Summary Human ₂macroglobulin combines two unique features: the non-active site directed inhibition of virtually all endoproteases and the selective clearance of ₂M-endoprotease complexes by receptor-mediated endocytosis. To study the molecular details of the mechanisms involved, primary amines were found to be worthwhile probes at three specific levels: the inactivation of native ₂M, the derivatization by factor XIII and the cellular process of receptor-recycling. In this paper published data are supplemented with recently obtained evidence to discuss and speculate on the possible action or involvement of transglutaminase activities, indicated by the effects of the primary amines.     

Crystal Structure of the α Subunit of Human Translation Initiation Factor 2B

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the α subunit of human eIF2B at 2.65 Å resolution. The eIF2Bα structure consists of the N-terminal α-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 Å in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 Å in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Bα. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Bα. Interestingly, the yeast eIF2Bα missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Bα missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The β and δ subunits of eIF2B are homologous to eIF2Bα and may have tertiary structures similar to the present eIF2Bα structure. The sulfate-ion-binding residues of eIF2Bα are well conserved in eIF2Bβ/δ. The abovementioned yeast and human missense mutations of eIF2Bβ/δ were also mapped on the eIF2Bα structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.     

X-Linkage of Human α-Galactosidase

A deficiency in α-galactosidase (α-Gal) activity–as measured aspecifically with the use of artificial substrates–is a regular feature in leucocytes and fibroblasts of patients affected by angiokeratoma corporis diffusum or Fabry's disease, a well-known X-linked trait in man^1–4. Fibroblast clones derived from mothers of affected males exhibit either normal or deficient activity of α-galactosidase. This demonstrates that the deficiency of α-galactosidase is caused by an X-linked mutation, but does not necessarily prove that the structural locus for this enzyme is itself located on the X-chromosome.     

Dysregulation of Human β-Defensin-2 Protein in Inflammatory Bowel Disease

Background

Human β-defensin-2 (HBD2) is an antimicrobial peptide implicated in the pathogenesis of inflammatory bowel disease (IBD). Low copy number and concomitant low mRNA expression of the HBD2 gene have been implicated in susceptibility to colonic Crohn''s Disease (CD). We investigated the colonic distribution of HBD2 mRNA expression, and the contributions of genetic and environmental factors on HBD2 protein production.

Methodology/Principal Findings

We examined HBD2 mRNA expression at three colonic locations by microarray analysis of biopsies from 151 patients (53 CD, 67 ulcerative colitis [UC], 31 controls). We investigated environmental and genetic influences on HBD2 protein production using ex vivo cultured sigmoid colon biopsies from 69 patients (22 CD, 26 UC, 21 controls) stimulated with lipopolysaccharide (LPS) and/or nicotine for 24 hours. HBD2 and cytokines were measured in culture supernatants. Using DNA samples from these patients, regions in the HBD2 gene promoter were sequenced for NF-κB binding-sites and HBD2 gene copy number was determined. HBD2 mRNA expression was highest in inflamed (vs. uninflamed p = 0.0122) ascending colon in CD and in inflamed (vs. uninflamed p<0.0001) sigmoid colon in UC. HBD2 protein production was increased in inflamed UC biopsies (p = 0.0078). There was no difference in HBD2 protein production from unstimulated biopsies of CD, UC and controls. LPS-induced HBD2 production was significantly increased in CD (p = 0.0375) but not UC (p = 0.2017); this LPS-induced response was augmented by nicotine in UC (p = 0.0308) but not CD (p = 0.6872). Nicotine alone did not affect HBD2 production. HBD2 production correlated with IL8 production in UC (p<0.001) and with IL10 in CD (p<0.05). Variations in the HBD2 promoter and HBD2 gene copy number did not affect HBD2 production.

Significance/Conclusions

Colonic HBD2 was dysregulated at mRNA and protein level in IBD. Inflammatory status and stimulus but not germline variations influenced these changes.     

Novel Antibacterial Activity of β2-Microglobulin in Human Amniotic Fluid

An antibacterial protein (about 12 kDa) was isolated from human amniotic fluid through dialysis, ultrafiltration and C18 reversed-phase HPLC steps. Automated Edman degradation showed that the N-terminal sequence of the antibacterial protein was NH₂-Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-. The N-terminal sequence of the antibacterial protein was found to be identical to that of β₂-microglobulin, a component of MHC class I molecules, which are present on all nucleated cells. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) revealed that the molecular mass of the antibacterial protein was 11,631 Da. This antibacterial protein, β₂M, possessed potent antibacterial activity against pathogenic bacteria. Specially, antibacterial activity was observed in potassium buffer, and potassium ion was found to be critical for the antibacterial activity. Interestingly, the antibacterial action of β₂M was associated with dissipation of the transmembrane potential, but the protein did not cause damage to the membrane that would result in SYTOX green uptake. In addition, stimulation of WISH amniotic epithelial cells with the bacterial endotoxin lipopolysaccharide (LPS) induced dose-dependent upregulation of β₂M mRNA expression. These results suggest that β₂M contributes to a self-defense response when amniotic cells are exposed to pathogens.     

Antimicrobial Activity of Inducible Human β Defensin-2 Against Mycoplasma pneumoniae

Defensins in innate immunity are known to play critical roles to protect the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between defensins and mycoplasmas. Human β defensin (hBD)-2 and hBD-3, but not hBD-1, were found to exert strikingly antimicrobial activity against Mycoplasma pneumoniae. To elucidate the role of defensins in M. pneumoniae infection, a human pulmonary squamous cell line EBC-1 was stimulated with M. pneumoniae and interleukin (IL)-1β. hBD-2 was markedly upregulated by IL-1β as well as M. pneumoniae, but neither hBD-1 nor hBD-3 was apparently upregulated. Thus, the results suggest that inducible hBD-2 would play a critical role in the protection of M. pneumoniae infection.     

2′,2′-Difluorodeoxycytidine Metabolism and Mechanism of Action In Human Leukemia Cells

Abstract

The toxicity of 2′,2′-difluorodeoxycytidine is due to the inhibition of DNA synthesis by a nucleotide metabolite by either direct inhibition of the process of DNA synthesis and/or to inhibition of ribonucleotide reductase.     

Isolation and Characterization of 2′-amino-modified RNA Aptamersfor Human TNFα

Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFα.Aptamers were selected from a starting pool of 40 randomized sequences composed of about 10^15 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFα and were further modified by replacement of 2′-OH with 2′-F and 2′-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFα was confirmed, and their activity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Results demonstrated that four 2′-NH2-modified aptamers bound to hTNFα with high affinity and blocked the binding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα. Oligonucleotide aptamers described here are potential therapeutics and diagnostics for hTNFα-related diseases.     

Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα

Human tumor necrosis factor a (hTNFa), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFa. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 1015 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFa and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFa was confirmed, and their activity to inhibit the cytotoxicity of hTNFa on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFa with high affinity and blocked the     

In Silico Modeling of Human α2C-Adrenoreceptor Interaction with Filamin-2

Vascular smooth muscle α_2C-adrenoceptors (α_2C-ARs) mediate vasoconstriction of small blood vessels, especially arterioles. Studies of endogenous receptors in human arteriolar smooth muscle cells (referred to as microVSM) and transiently transfected receptors in heterologous HEK293 cells show that the α_2C-ARs are perinuclear receptors that translocate to the cell surface under cellular stress and elicit a biological response. Recent studies in microVSM unraveled a crucial role of Rap1A-Rho-ROCK-F-actin pathways in receptor translocation, and identified protein-protein interaction of α_2C-ARs with the actin binding protein filamin-2 as an essential step in the process. To better understand the molecular nature and specificity of this interaction, in this study, we constructed comparative models of human α_2C-AR and human filamin-2 proteins. Finally, we performed in silico protein-protein docking to provide a structural platform for the investigation of human α_2C-AR and filamin-2 interactions. We found that electrostatic interactions seem to play a key role in this complex formation which manifests in interactions between the C-terminal arginines of α_2C-ARs (particularly R454 and R456) and negatively charged residues from filamin-2 region between residues 1979 and 2206. Phylogenetic and sequence analysis showed that these interactions have evolved in warm-blooded animals.     

Keratinocyte-Targeted Expression of Human Laminin γ2 Rescues Skin Blistering and Early Lethality of Laminin γ2 Deficient Mice

Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of “humanized” laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.     

Specific-sized Hyaluronan Fragments Promote Expression of Human β-Defensin 2 in Intestinal Epithelium

Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human β-defensin 2 (HβD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HβD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HβD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HβD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HβD2 protein.     

5′-Substituted 2′-Deoxycytidines as Non-Substrate Inhibitors of Human Deoxycytidine Kinase

Abstract

Several 5′-substituted analogues of 2′-deoxycytidine (dC), including 5′-amino2′,5′-dideoxycytidine (5′-amino-ddC), 5′-azido-2′,5′-dideoxycytidine (5′-azido-ddC) and 5′-O-ethyl-2′-deoxycytidine (5′-O-ethyl-dC), are non-substrate inhibitors of human dC kinase. Whereas 5′-amino-ddC inhibits phosphorylation of deoxyadenosine (dA) with K_i ～ 1 μ inhibition of phosphorylation of dC is bimodal and more effective at low inhibitor concentrations. In particular 5′-O-ethyl-dC inhibits phosphorylation of dA 10-fold more effectively (K_i ～ 3 μ) than that of dC (K_i ～ 30 μ). For 5′-azido-ddC inhibition was shown directly to be competitive with respect to substrate.     

β3-Adrenergic Regulation of L-Type Ca2+ Current and Force of Contraction in Human Ventricle

β₃-Adrenergic receptor (β₃-AR) is expressed in human atrial and ventricular tissues. Recently, we have demonstrated that it was involved in the activation of L-type Ca²⁺ current (I _Ca,L) in human atrial myocytes and the force of contraction of human atrial trabeculae. In the present study, we examined the effect of β₃-AR agonist CGP12177 which also is a β₁-AR/β₂-AR antagonist on I _Ca,L in human ventricular myocytes (HVMs) and the force of contraction of human ventricular trabeculae. CGP12177 stimulated I _Ca,L in HVMs with high potency but much lower efficacy than isoprenaline. The β₃-AR antagonist L-748,337 inhibited the effect of CGP12177. CGP12177 and L748,337 competed selectively on β₃-ARs because L748,337 had no effect on isoprenaline-induced stimulation of I _Ca,L, while CGP12177 completely blocked the effect of isoprenaline. The activation of β₃-ARs by CGP12177 does not involve the activation of G_i proteins because CGP12177 had no effect on forskolin-induced stimulation of I _Ca,L. CGP12177 had no effect on the force of contraction of human ventricular trabeculae. L-NMMA, an inhibitor of NO synthase, and IBMX, a nonselective inhibitor of phosphodiesterases, did not potentiate the effect of CGP12177 either on contraction of human ventricular trabeculae or on I _Ca,L in HVMs. We conclude that in human ventricles β₃-AR activation has no inotropic effect, while it slightly increases I _Ca,L. In contrast to human atrium, the activation of β₃-ARs in human ventricle is not accompanied by increased activity of phosphodiesterases.     

Molecular Insights into the Dynamics of Pharmacogenetically Important N-Terminal Variants of the Human β2-Adrenergic Receptor

The human β₂-adrenergic receptor (β₂AR), a member of the G-protein coupled receptor (GPCR) family, is expressed in bronchial smooth muscle cells. Upon activation by agonists, β₂AR causes bronchodilation and relief in asthma patients. The N-terminal polymorphism of β₂AR at the 16^th position, Arg16Gly, has warranted a lot of attention since it is linked to variations in response to albuterol (agonist) treatment. Although the β₂AR is one of the well-studied GPCRs, the N-terminus which harbors this mutation, is absent in all available experimental structures. The goal of this work was to study the molecular level differences between the N-terminal variants using structural modeling and atomistic molecular dynamics simulations. Our simulations reveal that the N-terminal region of the Arg variant shows greater dynamics than the Gly variant, leading to differential placement. Further, the position and dynamics of the N-terminal region, further, affects the ligand binding-site accessibility. Interestingly, long-range effects are also seen at the ligand binding site, which is marginally larger in the Gly as compared to the Arg variant resulting in the preferential docking of albuterol to the Gly variant. This study thus reveals key differences between the variants providing a molecular framework towards understanding the variable drug response in asthma patients.     

Characterization of the ��-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.