Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors

Homomeric alpha7 and heteromeric alpha4beta2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha7 receptors for which the alpha7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.     

Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.     

Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains

Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4+ T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4+ T cells and primary CD4+ T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs.     

Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.     

Topologies of a substrate protein bound to the chaperonin GroEL

The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.     

Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes

BACKGROUND: Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective transduction can be achieved in quiescent hepatocytes, by using virus at high titre. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. METHODS: We used two late-generation HIV-based lentiviral vector systems (pHR-SIN-cppT SGW and pRRLSIN.cPPT.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /- growth factors, hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations. RESULTS: Gene expression following lentiviral transduction of human liver cells in vitro was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transduced hepatocytes remained identical, but cells expressed more GFP protein. CONCLUSIONS: This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOIs, and reducing both cost and risk of viral-mediated toxicity.     

Melanosome maturation defect in Rab38-deficient retinal pigment epithelium results in instability of immature melanosomes during transient melanogenesis

Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the "chocolate" (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases.     

Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability

Most sporadic colorectal tumors carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein is involved in many processes that govern gut tissue. In addition to its involvement in the regulation of beta-catenin, APC is a cytoskeletal regulator with direct and indirect effects on microtubules. Cancer-related truncation mutations lack direct and indirect binding sites for microtubules in APC, suggesting that loss of this function contributes to defects in APC-mutant cells. In this study, we show that loss of APC results in disappearance of cellular protrusions and decreased cell migration. These changes are accompanied by a decrease in overall microtubule stability and also by a decrease in posttranslationally modified microtubules in the cell periphery particularly the migrating edge. Consistent with the ability of APC to affect cell shape, the overexpression of APC in cells can induce cellular protrusions. These data demonstrate that cell migration and microtubule stability are linked to APC status, thereby revealing a weakness in APC-deficient cells with potential therapeutic implications.     

Repression of alpha-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract

Polypyrimidine tract binding protein (PTB) acts as a regulatory repressor of a large number of alternatively spliced exons, often requiring multiple binding sites in order to repress splicing. In one case, cooperative binding of PTB has been shown to accompany repression. The SM exon of the alpha-actinin pre-mRNA is also repressed by PTB, leading to inclusion of the alternative upstream NM exon. The SM exon has a distant branch point located 386 nt upstream of the exon with an adjacent 26 nucleotide pyrimidine tract. Here we have analyzed PTB binding to the NM and SM exon region of the alpha-actinin pre-mRNA. We find that three regions of the intron bind PTB, including the 3' end of the polypyrimidine tract (PPT) and two additional regions between the PPT and the SM exon. The downstream PTB binding sites are essential for full repression and promote binding of PTB to the PPT with a consequent reduction in U2AF(65) binding. Our results are consistent with a repressive mechanism in which cooperative binding of PTB to the PPT competes with binding of U2AF(65), thereby specifically blocking splicing of the SM exon.     

Differential expression of the keratan sulphate proteoglycan,keratocan, during chick corneal embryogenesis

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the connective tissue matrix of the cornea of the eye, where they are believed to have functional roles in tissue organisation and transparency. Keratocan, is one of the three KS PGs expressed in cornea, and is the only one that is primarily cornea-specific. Work with the developing chick has shown that mRNA for keratocan is present in early corneal embryogenesis, but there is no evidence of protein synthesis and matrix deposition. Here, we investigate the tissue distribution of keratocan in the developing chick cornea as it becomes compacted and transparent in the later stages of development. Indirect immunofluorescence using a new monoclonal antibody (KER-1) which recognises a protein epitope on the keratocan core protein demonstrated that keratocan was present at all stages investigated (E10–E18), with distinct differences in localisation and organisation observed between early and later stages. Until E13, keratocan appeared both cell-associated and in the stromal extracellular matrix, and was particularly concentrated in superficial tissue regions. By E14 when the cornea begins to become transparent, keratocan was located in elongate arrays, presumably associated along collagen fibrils in the stroma. This fibrillar label was still concentrated in the anterior stroma, and persisted through E15–E18. Presumptive Bowman’s layer was evident as an unlabelled subepithelial zone at all stages. Thus, in embryonic chick cornea, keratocan, in common with sulphated KS chains in the E12–E14 developmental period, exhibits a preferential distribution in the anterior stroma. It undergoes a striking reorganisation of structure and distribution consistent with a role in relation to stromal compaction and corneal transparency. E. Claire Gealy and Briedgeen C. Kerr were joint first authors.