Proteomic and N-Terminomic TAILS Analyses of Human Alveolar Bone Proteins: Improved Protein Extraction Methodology and LysargiNase Digestion Strategies Increase Proteome Coverage and Missing Protein Identification.

With 2129 proteins still classified by the Human Proteome Organisation Human Proteome Project (HPP) as "missing" without compelling evidence of protein existence (PE) in humans, we hypothesized that in-depth proteomic characterization of tissues that are technically challenging to access and extract would yield evidence for tissue-specific missing proteins. Paradoxically, although the skeleton is the most massive tissue system in humans, as one of the poorest characterized by proteomics, bone falls under the HPP umbrella term as a "rare tissue". Therefore, we aimed to optimize mineralized tissue protein extraction methodology and workflows for proteomic and data analyses of small quantities of healthy young adult human alveolar bone. Osteoid was solubilized by GuHCl extraction, with hydroxyapatite-bound proteins then released by ethylenediaminetetraacetic acid demineralization. A subsequent GuHCl solubilization extraction was followed by solid-phase digestion of the remaining insoluble cross-linked protein using trypsin and then 6 M urea dissolution incorporating LysC digestion. Bone extracts were digested in parallel using trypsin, LysargiNase, AspN, or GluC prior to liquid chromatography-mass spectrometry analysis. Terminal Amine Isotopic Labeling of Substrates was used to purify semitryptic peptides, identifying natural and proteolytic-cleaved neo N-termini of bone proteins. Our strategy enabled complete solubilization of the organic bone matrix leading to extensive categorization of bone proteins in different bone matrix extracts, and hence matrix compartments, for the first time. Moreover, this led to the high confidence identification of pannexin-3, a "missing protein", found only in the insoluble collagenous matrix and revealed for the first time by trypsin solid-phase digestion. We also found a singleton proteotypic peptide of another missing protein, meiosis inhibitor protein 1. We also identified 17 proteins classified in neXtprot as PE1 based on evidence other than from MS, termed non-MS PE1 proteins, including ≥9-mer proteotypic peptides of four proteins.

Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability.

Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP(3) transfer. These mutations do not affect Cx26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating.

Connexin channels and phospholipids: association and modulation.

BACKGROUND: For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood. RESULTS: Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred. CONCLUSION: This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.

Structural studies of the N-terminus of Connexin 32 using 1H NMR spectroscopy.

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of a representative connexin molecule contains a flexible turn around glycine 12 [P.E. Purnick, D.C. Benjamin, V.K. Verselis, T.A. Bargiello, T.L. Dowd, Arch. Biochem. Biophys. 381 (2000) 181-190] allowing the N-terminus to reside at the cytoplasmic entry of the channel forming a voltage-sensor. Previous functional studies or neuropathies have shown that the mutation G12Y and G12S form non-functional channels while functional channels are formed from G12P. Using 2D (1)H NMR we show that similar to G12, the structure of the G12P mutant contains a more flexible turn around residue 12, whereas the G12S and G12Y mutants contain tighter, helical turns in this region. These results suggest an unconstrained turn is required around residue 12 to position the N-terminus within the pore allowing the formation of the cytoplasmic channel vestibule, which appears to be critical for proper channel function.

Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

An innexin-dependent cell network establishes left-right neuronal asymmetry in C. elegans.

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.

Innexins in the lobster stomatogastric nervous system: cloning, phylogenetic analysis, developmental changes and expression within adult identified dye and electrically coupled neurons.

Gap junctions play a key role in the operation of neuronal networks by enabling direct electrical and metabolic communication between neurons. Suitable models to investigate their role in network operation and plasticity are invertebrate motor networks, which are built of comparatively few identified neurons, and can be examined throughout development; an excellent example is the lobster stomatogastric nervous system. In invertebrates, gap junctions are formed by proteins that belong to the innexin family. Here, we report the first molecular characterization of two crustacean innexins: the lobster Homarus gammarus innexin 1 (Hg-inx1) and 2 (Hg-inx2). Phylogenetic analysis reveals that innexin gene duplication occurred within the arthropod clade before the separation of insect and crustacean lineages. Using in situ hybridization, we find that each innexin is expressed within the adult and developing lobster stomatogastric nervous system and undergoes a marked down-regulation throughout development within the stomatogastric ganglion (STG). The number of innexin expressing neurons is significantly higher in the embryo than in the adult. By combining in situ hybridization, dye and electrical coupling experiments on identified neurons, we demonstrate that adult neurons that express at least one innexin are dye and electrically coupled with at least one other STG neuron. Finally, two STG neurons display no detectable amount of either innexin mRNAs but may express weak electrical coupling with other STG neurons, suggesting the existence of other forms of innexins. Altogether, we provide evidence that innexins are expressed within small neuronal networks built of dye and electrically coupled neurons and may be developmentally regulated.

Paciente con enfermedad corneal severa en el contexto del síndrome KID / Patient with severe corneal disease in KID syndrome

Caso clínico: Mujer de 33 años con neovascularizacióncorneal bilateral superficial y profunda yqueratopatía punteada superficial de distribucióndifusa, queratoeritema y sordera neurosensorial,que es diagnosticada de síndrome KID.Discusión: El síndrome KID es una displasia congénitaectodérmica caracterizada por la asociaciónde queratitis vascularizante, lesiones cutáneashiperqueratósicas y sordera neurosensorial. Recientemente,la deficiencia de stem cell limbares ha sidoreconocida como posible factor patogenético clave

Case report: A 33-year-old woman with superficial ;;and deep bilateral corneal vascularization and ;;keratoconjunctivitis sicca, keratoerythema and neurosensory ;;deafness, was diagnosed with keratitisichthyosis- ;;deafness (KID) syndrome. ;;Discussion: KID syndrome is a congenital ectodermal ;;dysplasia characterized by the association of ;;vascularizing keratitis, hyperkeratotic skin lesions ;;and sensorineural hearing loss. Recently, limbal ;;stem cell deficiency was recognized as a possible ;;major pathogenetic factor

Emerging complexities in identity and function of glial connexins.

Recent research results indicate that glial gap-junction communication is much more complex and widespread than originally thought, and has diverse roles in brain homeostasis and the response of the brain to injury. The situation is far from clear, however. Pharmacological agents that block gap junctions can abolish neuron-glia long-range signaling and can alleviate neuronal damage whereas, intriguingly, opposite effects are observed in mice lacking connexin43, a major gap-junction subunit protein in astrocytes. How can the apparently contradictory results be explained, and how is specificity achieved within the glial gap-junction system? Another key issue in understanding glial connexin function is that oligodendrocytes and astrocytes, each of which express distinct connexin isotypes, are thought to participate in brain homeostasis by forming a panglial syncytium. Molecular analysis has revealed a surprising diversity of connexin expression and function, and this has led to new hypotheses regarding their roles in the brain, which could be tested using new approaches.

Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells.

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10-20 microg of pure connexin protein from 2.5x10(8) HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)-agarose make this affinity tagging and purification procedure easily applicable to other proteins.

Isolation and characterization of gap junctions from tissue culture cells.

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.

Differential expression of gap-junction gene connexin 31 in seminiferous epithelium of rat testes.

Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of certain gap-junction connexins. Our findings by RT-PCR indicate that the Cx31 gene is expressed in testis tissue of adult and postnatal rats. During the postnatal spermatogenic process, the Cx31-specific signal became detectable at 15 dpp and onward by in situ hybridization, and apparently localized in the basal compartment of seminiferous epithelium where active spermatogonia and early primary spermatocytes reside. No signal was found in the luminal region. In adult testes, spermatids of elongation phase were also Cx31 positive. Immunohistochemical analysis with mouse anti-Cx31 antibody gave a similar staining pattern, providing further evidence that the gap-junction protein is abundant in the basal seminiferous epithelium, in accordance with the cellular distribution of Cx31 mRNA. These results represent the first demonstration of Cx31 expression at both transcriptional and protein levels in the seminiferous epithelium of rat testes. Thus, Cx31 may play a role in cell-cell communication during spermatogenesis.

Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells.

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.

The gap-junction protein connexin 56 is phosphorylated in the intracellular loop and the carboxy-terminal region.

The lens gap-junction protein, connexin 56, is modified by phosphorylation. Two-dimensional mapping of tryptic phosphopeptides of 32P-labeled connexin 56 from primary chicken-lens cultures showed that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) induced an increase in phosphorylation of connexin 56 at specific constitutively phosphorylated sites. Treatment with 8-Br-cAMP or forskolin did not induce substantial changes in connexin 56 phosphorylation. Two phosphorylation sites within connexin 56, S493 and S118, were identified after HPLC purification and peptide sequencing of tryptic phosphopeptides from bacterially expressed connexin 56 fusion proteins phosphorylated by protein kinase C or protein kinase A in vitro. Comparisons of the two-dimensional maps of tryptic phosphopeptides from in vitro phosphorylated connexin 56 fusion proteins and in vivo phosphorylated connexin 56 showed that S493 and S118 were constitutively phosphorylated in lentoid-containing cultures, and that treatment with TPA induced an increase in phosphorylation of the peptides containing S118. It is suggested that phosphorylation of connexin 56 at S118 is involved in the TPA-induced decrease in intercellular communication and acceleration of connexin 56 degradation.

Channel-forming activity of immunoaffinity-purified connexin32 in single phospholipid membranes.

Connexin32, a member of the family of proteins that forms gap junction channels between cells, was immunoaffinity-purified from rat liver using a monoclonal antibody, under nondenaturing conditions and reconstituted into unilamellar phospholipid liposomes and bilayers. Gel-filtration studies indicate that the connexin32 is purified predominantly in structures of a size consistent with that of single hemichannels and too small to be junctional channels (dimers of hemichannels). Purified connexin formed channels permeable to sucrose and to Lucifer Yellow. The permeability was reversibly reduced by acidic pH and unaffected by several agents that modulate coupling between cells. Modeling of the distribution of the permeability in the liposomes indicates that it is mediated by connexin structures that distribute among the liposomes as single hemichannels. Bilayer recordings of the purified connexin show high conductance channels with asymmetric voltage sensitivity. The results show that immunopurified connexin32 can form channels, in single phospholipid membranes, that have permeability similar to that of gap junction channels and thus can be utilized in studies of permeability and its regulation to investigate its role in normal physiological function, development, and disease.

Molecular cloning of sheep connexin49 and its identity with MP70.

The nucleotide sequence of the sheep homologue of the lens-specific mouse connexin50, chicken connexin45.6, and human connexin50 has been obtained following screening of a sheep genomic library. This connexin comprises 1323 nucleotides, coding for a protein of 440 amino acid residues and a predicted molecular weight of 49,160 daltons, so by convention is termed sheep connexin49. A connexin49 cDNA probe detected a single major band with a mobility of 6.8 kb in sheep lens RNA, but not in RNA isolated from five other sheep organs. The N-terminal amino acid sequence of sheep connexin49 is identical to that of mouse connexin50 and closely matches that of MP70, indicating the identity of sheep connexin49 with MP70. The nucleotide and translated amino acid sequences of connexin49 have 69-87% and 76%-87% identity respectively with chicken connexin45.6, human connexin50 and mouse connexin50. Like other members of this lens connexin family, sheep connexin49 coding region is completely contained within one exon, and the sequence of the N-terminal region, the four transmembrane domains and the two extracellular loops are highly conserved.