Antizyme Inhibitor 2 : A Novel Regulator of Cellular Polyamine Homeostasis

Polyamines are organic polycations that participate in various physiological functions, including cell proliferation, differentiation and apoptosis. Cellular polyamines originate from endogenous biosynthesis and exogenous sources. Their subcellular pool is under strict control, achieved by regulating their uptake and metabolism. Polyamine-induced proteins called antizymes (AZ) act as key regulators of intracellular polyamine concentration. They regulate both the transport of polyamines and the activity and degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. AZs themselves are negatively regulated by antizyme inhibitor (AZIN). AZIN functions as a positive regulator of cellular polyamine homeostasis, which by binding to AZs reactivates ODC and induces the uptake of polyamines. In various pathological conditions, including cancer, polyamine levels are misregulated. Polyamine homeostasis has therefore become an attractive target for therapeutic interventions and it is thus crucial to characterize the molecular basis underlying the homeostatic regulation. A novel human AZIN-resembling protein was previously identified in our group. The purpose of this study was to elucidate the function and distribution of this protein, termed as an antizyme inhibitor 2 (AZIN2). According to my results, AZIN2 functions as a novel regulator of polyamine homeostasis. It shows no enzymatic activity, but instead it binds AZs and negates their activity, which subsequently leads to reactivation of ODC and inhibition of its degradation. Expression of AZIN2 is restricted to terminally differentiated cells, such as mast cells (MC) and neurosecretory cells. In these actively secreting cell types, AZIN2 localizes to subcellular vesicles or granules where its function is important for the vesicle-mediated secretion. In MCs, AZIN2 localizes to the serotonin-containing subset of MC granules, and its expression is coupled to MC activation. The functional role of polyamines as potential mediators of MC activity was also investigated, and it was observed that the secretion of serotonin is selectively dependent on activation of ODC. In neurosecretory cells, AZIN2-positive vesicles localize mainly to the trans-Golgi network (TGN). Depletion of AZIN2 or cellular polyamines causes selective fragmentation of the TGN and retards secretion of proteins. Since addition of exogenous polyamines reverses these effects, the data indicate that AZIN2 and its downstream effectors, polyamines, are functionally implicated in the regulation of secretory vesicle transport. My studies therefore reveal a novel function for polyamines as modulators of both constitutive and regulated secretion. Based on the results, I propose that the role of AZIN2 is to act as a local in situ activator of polyamine biosynthesis.

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis Novel miniaturized mass spectrometric ionization techniques based on atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were studied and evaluated in the analysis of environmental samples and biosamples. The three analytical systems investigated here were gas chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry (GC-µAPCI-MS) and gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry (GC-µAPPI-MS), where sample pretreatment and chromatographic separation precede ionization, and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS), where the samples are analyzed either as such or after minimal pretreatment. The gas chromatography-microchip atmospheric pressure ionization-mass spectrometry (GC-µAPI-MS) instrumentations were used in the analysis of polychlorinated biphenyls (PCBs) in negative ion mode and 2-quinolinone-derived selective androgen receptor modulators (SARMs) in positive ion mode. The analytical characteristics (i.e., limits of detection, linear ranges, and repeatabilities) of the methods were evaluated with PCB standards and SARMs in urine. All methods showed good analytical characteristics and potential for quantitative environmental analysis or bioanalysis. Desorption and ionization mechanisms in DAPPI were studied. Desorption was found to be a thermal process, with the efficiency strongly depending on thermal conductivity of the sampling surface. Probably the size and polarity of the analyte also play a role. In positive ion mode, the ionization is dependent on the ionization energy and proton affinity of the analyte and the spray solvent, while in negative ion mode the ionization mechanism is determined by the electron affinity and gas-phase acidity of the analyte and the spray solvent. DAPPI-MS was tested in the fast screening analysis of environmental, food, and forensic samples, and the results demonstrated the feasibility of DAPPI-MS for rapid screening analysis of authentic samples.

Comprehensive Two-Dimensional Gas Chromatography: Instrumental and Methodological Development

Comprehensive two-dimensional gas chromatography (GC×GC) offers enhanced separation efficiency, reliability in qualitative and quantitative analysis, capability to detect low quantities, and information on the whole sample and its components. These features are essential in the analysis of complex samples, in which the number of compounds may be large or the analytes of interest are present at trace level. This study involved the development of instrumentation, data analysis programs and methodologies for GC×GC and their application in studies on qualitative and quantitative aspects of GC×GC analysis. Environmental samples were used as model samples. Instrumental development comprised the construction of three versions of a semi-rotating cryogenic modulator in which modulation was based on two-step cryogenic trapping with continuously flowing carbon dioxide as coolant. Two-step trapping was achieved by rotating the nozzle spraying the carbon dioxide with a motor. The fastest rotation and highest modulation frequency were achieved with a permanent magnetic motor, and modulation was most accurate when the motor was controlled with a microcontroller containing a quartz crystal. Heated wire resistors were unnecessary for the desorption step when liquid carbon dioxide was used as coolant. With use of the modulators developed in this study, the narrowest peaks were 75 ms at base. Three data analysis programs were developed allowing basic, comparison and identification operations. Basic operations enabled the visualisation of two-dimensional plots and the determination of retention times, peak heights and volumes. The overlaying feature in the comparison program allowed easy comparison of 2D plots. An automated identification procedure based on mass spectra and retention parameters allowed the qualitative analysis of data obtained by GC×GC and time-of-flight mass spectrometry. In the methodological development, sample preparation (extraction and clean-up) and GC×GC methods were developed for the analysis of atmospheric aerosol and sediment samples. Dynamic sonication assisted extraction was well suited for atmospheric aerosols collected on a filter. A clean-up procedure utilising normal phase liquid chromatography with ultra violet detection worked well in the removal of aliphatic hydrocarbons from a sediment extract. GC×GC with flame ionisation detection or quadrupole mass spectrometry provided good reliability in the qualitative analysis of target analytes. However, GC×GC with time-of-flight mass spectrometry was needed in the analysis of unknowns. The automated identification procedure that was developed was efficient in the analysis of large data files, but manual search and analyst knowledge are invaluable as well. Quantitative analysis was examined in terms of calibration procedures and the effect of matrix compounds on GC×GC separation. In addition to calibration in GC×GC with summed peak areas or peak volumes, simplified area calibration based on normal GC signal can be used to quantify compounds in samples analysed by GC×GC so long as certain qualitative and quantitative prerequisites are met. In a study of the effect of matrix compounds on GC×GC separation, it was shown that quality of the separation of PAHs is not significantly disturbed by the amount of matrix and quantitativeness suffers only slightly in the presence of matrix and when the amount of target compounds is low. The benefits of GC×GC in the analysis of complex samples easily overcome some minor drawbacks of the technique. The developed instrumentation and methodologies performed well for environmental samples, but they could also be applied for other complex samples.

Heparin-binding growth-associated molecule (HB-GAM) in activity-dependent neuronal plasticity in hippocampus

Cell adhesion and extracellular matrix (ECM) molecules play a significant role in neuronal plasticity both during development and in the adult. Plastic changes in which ECM components are implicated may underlie important nervous system functions, such as memory formation and learning. Heparin-binding growthassociated molecule (HB-GAM, also known as pleiotrophin), is an ECM protein involved in neurite outgrowth, axonal guidance and synaptogenesis during perinatal period. In the adult brain HB-GAM expression is restricted to the regions which display pronounced synaptic plasticity (e.g., hippocampal CA3-CA1 areas, cerebral cortex laminae II-IV, olfactory bulb). Expression of HB-GAM is regulated in an activity-dependent manner and is also induced in response to neuronal injury. In this work mutant mice were used to study the in vivo function of HB-GAM and its receptor syndecan-3 in hippocampal synaptic plasticity and in hippocampus-dependent behavioral tasks. Phenotypic analysis of HBGAM null mutants and mice overexpressing HB-GAM revealed that opposite genetic manipulations result in reverse changes in synaptic plasticity as well as behavior in the mutants. Electrophysiological recordings showed that mice lacking HB-GAM have an increased level of long-term potentiation (LTP) in the area CA1 of hippocampus and impaired spatial learning, whereas animals with enhanced level of HB-GAM expression have attenuated LTP, but outperformed their wild-type controls in spatial learning. It was also found that GABA(A) receptor-mediated synaptic transmission is altered in the transgenic mice overexpressing HB-GAM. The results suggest that these animals have accentuated hippocampal GABAergic inhibition, which may contribute to the altered glutamatergic synaptic plasticity. Structural studies of HB-GAM demonstrated that this protein belongs to the thrombospondin type I repeat (TSR) superfamily and contains two β-sheet domains connected by a flexible linker. It was found that didomain structure is necessary for biological activity of HB-GAM and electrophysiological phenotype displayed by the HB-GAM mutants. The individual domains displayed weaker binding to heparan sulfate and failed to promote neurite outgrowth as well as affect hippocampal LTP. Effects of HB-GAM on hippocampal synaptic plasticity are believed to be mediated by one of its (co-)receptor molecules, namely syndecan-3. In support of that, HB-GAM did not attenuate LTP in mice deficient in syndecan-3 as it did in wild-type controls. In addition, syndecan-3 knockout mice displayed electrophysiological and behavioral phenotype similar to that of HB-GAM knockouts (i.e. enhanced LTP and impaired learning in Morris water-maze). Thus HB-GAM and syndecan-3 are important modulators of synaptic plasticity in hippocampus and play a role in regulation of learning-related behavior.

Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

Nikotiinireseptoreiden kautta vaikuttavat lääkeaineet nikotiiniriippuvuuden ja neurologisten sairauksien hoidossa

Neuronaaliset nikotiinireseptorit liittyvät tupakkariippuvuuden lisäksi moniin neurologisiin sairauksiin, kuten Alzheimerin tautiin, skitsofreniaan, masennukseen ja tarkkaavaisuus- ja ylivilkkaushäiriöön. Nikotiinireseptorien stimulaation on tutkimuksissa havaittu parantavan kognitiota. Useat lääkeyritykset tutkivat nikotiinireseptoriagonisteja ja -antagonisteja eri neurologisten sairauksien hoidossa. Ongelmana nikotiinireseptori-agonisteja käytettäessä on reseptorissa tapahtuva desensitisaatio. Tällöin reseptori sulkeutuu, eikä aktivoidu vaikka agonistia olisi tarjolla tai sitoutuneena reseptoriin. Varsinkin alfa7-reseptori desensitoituu hyvin nopeasti agonistialtistuksen seurauksena. Reseptorien desensitoituminen voi kliinisessä käytössä aiheuttaa lääkeaineen tehon menetyksen. Perinteisen agonistin sitoutumiskohdan lisäksi nikotiinireseptorissa sijaitsee myös muita sitoutumiskohtia, joita kutsutaan allosteerisiksi sitoutumispaikoiksi. Tutkimuksissa on havaittu, että eräät allosteerisesti sitoutuvat aineet, kuten PNU-120596, voivat vahvistaa agonistin aikaansaamaa vastetta ja/tai estää reseptorin desensitoitumista. Näitä aineita kutsutaan positiivisiksi allosteerisiksi modulaattoreiksi ja niiden ajatellaan olevan vaihtoehto desensitoitumisen aiheuttamaan tehon menetyksen ongelmaan. Nikotiinireseptorien positiivisten allosteeristen modulaattorien tarkkaa vaikutusta ja sitoutumiskohtaa reseptoriin ei vielä tarkkaan tiedetä. Tutkimuksen aiheena oli karakterisoida positiivisten allosteeristen modulaattoreiden vaikutuksia alfa7-nikotiinireseptoriin. Tutkimuksessa tarkoituksena oli käyttää hyväksi laboratoriossa aiemmin tehtyä havaintoa, jonka mukaan alfa7-nikotiinireseptorin transmembraaniosan aminohappoon tehdyn mutaation L247T seurauksena positiiviset allosteeriset modulaattorit muuttuvat agonisteiksi. Haluttiin selvittää, kuinka agonistin sitoutumiskohtaan kohdennettua mutageneesiä käyttäen tehty mutaatio W149M tai W149F vaikuttavat PNU-120596:n kykyyn toimia agonistina alfa7L247T reseptoriin. Asetyylikoliini toimi konventionaalisen agonistin mallina tutkimuksessa. Tutkimuksen toinen tavoite oli tehdä mutaatio M253Lalfa7-reseptorin transmembraaniosaan. Mutaation on todettu estävän allosteeristen potentiaattoreiden kykyä voimistaa agonistin aikaansaamaa vastetta. Tarkoitus oli tutkia millaisia vaikutuksia M253L-mutaatiolla on allosteerisen potentiaattorin kykyyn toimia agonistina L247T-mutaation sisältävään reseptoriin. Mutatoidun reseptorin mRNA mikroinjektoitiin oosyyttiin ja elektrofysiologian avulla tutkittiin ilmennettyjen reseptorien toimintaa käyttäen kahden elektrodin jännitelukitus -menetelmää. Kaikki suunnitellut mutaatiot saatiin tehtyä onnistuneesti alfa7- ja alfa7L247T-reseptoreihin. Ortosteerisen sitoutumiskohdan mutaatio villin tyypin Į7-reseptorissa vaikutti hyvin voimakkaasti joko asetyylikoliinin sitoutumiseen reseptoriin tai reseptorin toimintaan, sillä asetyylikoliinilla ei reseptorista saatu mitattua vasteita. Myöskään PNU-120596 yksinään ei saanut aikaan vasteita alfa7W149M-reseptorissa. Kaksoismutatoidussa alfa7W149M/L247T-reseptorissa puolestaan havaittiin, että asetyylikoliinin annos-vaste -kuvaaja siirtyi huomattavasti enemmän oikealle kuin PNU-120596:n, kun verrattiin annos-vaste –kuvaajia alfa7L247T ja alfa7W149M/L247T–reseptoreiden välillä. Transmembraaniosan mutaatio M253L ei vaikuttanut PNU-120596:n kykyyn toimia agonistina alfa7L247T-reseptoriin, eikä sillä ollut vaikutusta asetyylikoliinin annosvaste-kuvaajiin. Tutkimus tukee aiempia havaintoja siitä, että positiivisten allosteeristen modulaattoreiden sitoutumiskohta nikotiinireseptorissa sijaitsisi transmembraaniosassa. M253L-mutaation osalta tulokset ovat hieman ristiriidassa aiempien tulosten kanssa. L247T-mutaatio vaikuttaa hyvin voimakkaasti nikotiinireseptorin toimintaan sekä sijaitsee aminohapon M253 läheisyydessä. On mahdollista, että se peittää M253L-mutaation vaikutuksen. Toisaalta voi olla, että M253 on aminohappo, joka vaikuttaa vain reseptorivasteiden voimistumiseen eikä allosteeristen potentiaattoreiden sitoutumiseen.