Uncovering the functional constraints underlying the genomic organisation of the Odorant-Binding Protein genes

Animal olfactory systems have a critical role for the survival and reproduction of individuals. In insects, the odorant-binding proteins (OBPs) are encoded by a moderately sized gene family, and mediate the first steps of the olfactory processing. Most OBPs are organized in clusters of a few paralogs, which are conserved over time. Currently, the biological mechanism explaining the close physical proximity among OBPs is not yet established. Here, we conducted a comprehensive study aiming to gain insights into the mechanisms underlying the OBP genomic organization. We found that the OBP clusters are embedded within large conserved arrangements. These organizations also include other non-OBP genes, which often encode proteins integral to plasma membrane. Moreover, the conservation degree of such large clusters is related to the following: 1) the promoter architecture of the confined genes, 2) a characteristic transcriptional environment, and 3) the chromatin conformation of the chromosomal region. Our results suggest that chromatin domains may restrict the location of OBP genes to regions having the appropriate transcriptional environment, leading to the OBP cluster structure. However, the appropriate transcriptional environment for OBP and the other neighbor genes is not dominated by reduced levels of expression noise. Indeed, the stochastic fluctuations in the OBP transcript abundance may have a critical role in the combinatorial nature of the olfactory coding process.

Microtubule-associated protein 6 mediates neuronal connectivity through Semaphorin 3E-dependent signalling for axonal growth.

Structural microtubule associated proteins (MAPs) stabilize microtubules, a property that was thought to be essential for development, maintenance and function of neuronal circuits. However, deletion of the structural MAPs in mice does not lead to major neurodevelopment defects. Here we demonstrate a role for MAP6 in brain wiring that is independent of microtubule binding. We find that MAP6 deletion disrupts brain connectivity and is associated with a lack of post-commissural fornix fibres. MAP6 contributes to fornix development by regulating axonal elongation induced by Semaphorin 3E. We show that MAP6 acts downstream of receptor activation through a mechanism that requires a proline-rich domain distinct from its microtubule-stabilizing domains. We also show that MAP6 directly binds to SH3 domain proteins known to be involved in neurite extension and semaphorin function. We conclude that MAP6 is critical to interface guidance molecules with intracellular signalling effectors during the development of cerebral axon tracts.

The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins.

Role of Membrane Transporters, P-Glycoprotein and Mrp1, in The Placental Transfer of Drugs With Special Reference to Saquinavir and Quetiapine

Drug transporting membrane proteins are expressed in various human tissues and blood-tissue barriers, regulating the transfer of drugs, toxins and endogenous compounds into or out of the cells. Various in vitro and animal experiments suggest that P-glycoprotein (P-gp) forms a functional barrier between maternal and fetal blood circulation in the placenta thereby protecting the fetus from exposure to xenobiotics during pregnancy. The multidrug resistance-associated protein 1 (MRP1) is a relatively less studied transporter protein in the human placenta. The aim of this study series was to study the role of placental transporters, apical P-gp and basal MRP1, using saquinavir as a probe drug, and to study transfer of quetiapine and the role of P-gp in its transfer in the dually perfused human placenta/cotyledon. Furthermore, two ABCB1 (encoding P-gp) polymorphisms (c.3435C>T, p.Ile1145Ile and c.2677G>T/A, p.Ala893Ser/Thr) were studied to determine their impact on P-gp protein expression level and on the transfer of the study drugs. Also, the influence of the P-gp protein expression level on the transfer of the study drugs was addressed. Because P-gp and MRP1 are ATP-dependent drug-efflux pumps, it was studied whether exogenous ATP is needed for the function of ATP-dependent transporter in the present experimental model. The present results indicated that the addition of exogenous ATP was not necessary for transporter function in the perfused human placental cotyledon. Saquinavir and quetiapine were both found to cross the human placenta; transplacental transfer (TPTAUC %) for saquinavir was <0.5% and for quetiapine 3.7%. Pharmacologic blocking of P-gp led to disruption of the blood-placental barrier (BPB) and increased the placental transfer of P-gp substrate, saquinavir, into the fetal circulation by 6- to 8-fold. In reversed perfusions P-gp, MRP1 and possibly OATP2B1 had a negligible role in the fetal-to-maternal transfer of saquinavir. The TPTAUC % of saquinavir was about 100-fold greater from the fetal side to the maternal side compared with the maternal-to-fetal transfer. P-gp activity is not likely to modify the placental transfer of quetiapine. Higher P-gp protein expression levels were associated with the variant allele 3435T, but no correlation was found between the TPTAUC % of saquinavir and placental P-gp protein expression. The present results indicate that P-gp activity drastically affects the fetal exposure to saquinavir, and suggest that pharmacological blockade of the P-gp activity during pregnancy may pose an increased risk for adverse fetal outcome. The blockade of P-gp activity could be used in purpose to obtain higher drug concentration to the fetal side, for example, in prevention (to decrease virus transfer to fetal side) or in treating sick fetus.

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.

Amyloid Beta Precursor Protein: Proper Credit for the Basic Biochemical Properties of the Most Studied Protein in the 21st Century

The amyloid precursor protein (APP) is mainly known for being the precursor of the ß-amyloid peptide, which accumulates in plaques found in the brain of Alzheimer's disease patients. Expression in different tissues and the degree of sequence identity among mammals indicate an essential and non-tissue specific physiological function. APP is anchored to the membrane and displays a single C-terminal intracellular domain and a longer N-terminal extracellular domain. The basic biochemical properties and the scattered data on research, not related to production of beta-amyloid peptide, suggest that the protein and the molecules resulting from APP proteolytic cleavage may act as adhesion factors, enzymes, hormones/neurotransmitters and/or protease inhibitors. APP deserves to be known for its quite notable properties and its physiological role(s).

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

Sequence analysis of the capsid protein gene of an isolate of Apple stem grooving virus, and its survey in Southern Brazil

Apple stem grooving virus (ASGV) is one of the most important viruses infecting fruit trees. This study aimed at the molecular characterization of ASGV infecting apple (Malus domestica) plants in Santa Catarina (SC). RNA extracted from plants infected with isolate UV01 was used as a template for RT-PCR using specific primers. An amplified DNA fragment of 755 bp was sequenced. The coat protein gene of ASGV isolate UV01 contains 714 nucleotides, coding for a protein of 237 amino acids with a predicted Mr of approximately 27 kDa. The nucleotide and the deduced amino acid sequences of the coat protein gene showed identities of 90.9% and 97.9%, respectively, with a Japanese isolate of ASGV. Very high amino acid homologies (98.7%) were also found with Citrus tatter leaf capillovirus (CTLV), a very close relative of ASGV. These results indicate low coat protein gene variability among Capillovirus isolates from distinct regions. In a restricted survey, mother stocks in orchards and plants introduced into the country for large scale fruit production were indexed and shown to be infected by ASGV (20%), usually in a complex with other (latent) apple viruses (80%).

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.