Abstract Rheumatoid arthritis (RA) is an autoimmune disease causing inflammation of synovial joints, which may lead to permanent changes in cartilage and bone tissues. RA patients have antibodies binding to citrullinated and also to carbamylated proteins. Antibodies binding to citrulline are associated with more severe disease progression and may already appear years before clinical disease onset. Citrulline and the lysine carbamylation product, homocitrulline, are similar in structure. Citrullinated proteins have been studied in RA and in neurological diseases, but researchers have been unaware of the effect of homocitrulline in citrulline detection methods. The purpose of the present study was to clarify the features of protein-bound citrulline and homocitrulline in relation to research done on citrullination and in immunological reactions related to rheumatoid arthritis. In the first study of this thesis the confounding role of homocitrulline in citrulline detection was shown. In the first and second study the features of experimentally induced antibodies were assessed. The antibodies induced with citrulline- and homocitrulline-containing protein structures were shown to react both with the ureido groups and the protein structures. The antibodies were able to distinguish between citrulline and homocitrulline in the same sequence even though binding to both. In the third study the simultaneous presence of citrulline and homocitrulline in RA synovial tissue was shown. In conclusion, considering the simultaneous presence of citrulline and homocitrulline and the binding features of the experimentally induced antibodies, homocitrulline could have a yet unsolved role in RA. Secondly, the existence of homocitrulline should be borne in mind in studies on citrullination. / Tiivistelmä Nivelreuma on niveltulehduksen aiheuttava autoimmuunitauti, joka voi johtaa pysyviin muutoksiin nivelen rusto- ja luukudoksessa. Nivelreumaa sairastavilla esiintyy vasta-aineita sitrullinoituneita ja karbamyloituneita proteiineja vastaan. Sitrulliiniin sitoutuvia vasta-aineita voi esiintyä elimistössä jo vuosia ennen taudin puhkeamista, ja niiden esiintyminen on yhdistetty vaikeampaan taudinkuvaan. Sitrulliini ja lysiinin karbamylaatiotuote homositrulliini ovat rakenteellisesti samankaltaisia. Proteiinien sitrullinaatiota on tutkittu nivelreumassa ja neurologisissa taudeissa, mutta homositrulliinin olemassaoloa tai sen vaikutusta tutkimusmenetelmiin ei ole huomioitu. Tämän tutkimuksen tarkoituksena oli selvittää proteiineihin sitoutuneiden sitrulliinin ja homositrulliinin ominaisuuksia aikaisempiin tutkimuksiin ja nivelreuman immunologisiin reaktioihin liittyen. Tässä tutkimuksessa homositrulliinin osoitettiin häiritsevän sitrulliinin tunnistamista. Ensimmäisessä ja toisessa osatyössä aiheutettiin kokeellisesti vasta-aineita sitrulliinia ja homositrulliinia sisältävillä proteiinirakenteilla. Vasta-aineiden havaittiin reagoivan sekä ureidoryhmän että proteiinirakenteen kanssa. Vasta-aineet pystyivät erottamaan sitrulliinin ja homositrulliinin toisistaan samassa rakenteessa, vaikka sitoutuivat kumpaankin. Kolmannessa osatyössä osoitettiin, että sitrulliinia ja homositrulliinia esiintyy samanaikaisesti nivelreumapotilaan tulehtuneessa nivelkalvossa. Tutkimus osoitti, että sitrulliinin ja homositrulliinin samanaikainen esiintyminen ja kokeellisesti aiheutettujen vasta-aineiden ominaisuudet huomioiden homositrulliinilla voi olla jokin toistaiseksi selvittämätön rooli nivelreumassa. Homositrulliinin olemassaolo on syytä huomioida sitrullinaatiota tutkittaessa.

Innate immunity is the first line of defense against invading pathogens. It provides immediate protection by initiating both cellular and humoral immune reactions in response to a wide range of infections. It is also important to the development of long-lasting and pathogen-specific adaptive immunity. Thus, studying of the innate immunity, especially the pathogen recognition and signaling modulation, is crucial for understanding the intrinsic mechanisms underlying the host defense, as well as contributing the development of the fight against infectious diseases. Drosophila is an ideal model organism for study of innate immunity. Comparing to mammals, Drosophila immunity is relative conserved and less redundant. A variety of molecular and genetic tools available add further convenience to the research in this system. My work is focused on the signaling modulation by post-translational modification after activation. In these studies I demonstrated in the center of Imd pathway, the Imd protein undergoes proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination/degradation in a stimulation-dependent manner. These modifications of Imd play a crucial role in regulating signaling in response to infection. The characterization of ubiquitin-editing event provides a new insight into the molecular mechanisms underlying the activation and termination of insect immune signaling pathway.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (>170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis.

Abstract Cellular signaling by G protein-coupled receptors (GPCRs) governs a wide array of physiological functions throughout the body. The human δ-opioid receptor (hδOR) is a GPCR that modulates the sensation of pain and mood and has great potential for the treatment of pain and a variety of neurological disorders. A common single-nucleotide polymorphism (SNP) in the extracellular N-terminal tail of hδOR changes Phe to Cys at position 27. Using various biochemical and cell biological methods, the study demonstrates that several events during receptor biosynthesis and cell surface delivery are affected by the SNP. These events participate in the multifaceted regulation of the receptor and modulate receptor behavior at the cell surface. Two distinct pathways were shown to scrutinize the quality of the synthesized hδOR in the endoplasmic reticulum (ER) and target some for degradation in N-glycan-dependent and -independent ways. The hδORCys27 that matures inefficiently required N-glycan-mediated interactions with the lectin-chaperone calnexin to be expressed in a fully functional form at the cell surface, whereas the N-glycan-independent pathway was sufficient for hδORPhe27. For both variants, the N-glycan-independent quality control, which is likely to operate as a back-up pathway, led to a more rapid export from the ER and receptors at the cell surface that were less stable. Receptor dimerization emerged as an important regulatory step for receptor cell surface delivery. In co-transfected cells, interactions between the newly-synthesized variants led to the retention and subsequent ER-associated degradation of hδORPhe27. This dominant-negative attenuation of hδORPhe27 cell surface expression by hδORCys27 may have unpredictable consequences for opioid signaling in heterozygous individuals. Finally, the study shows that N-acetylgalactosamine (GalNAc)-type O-glycosylation catalyzed in the Golgi modulates hδOR expression at the cell surface by enhancing receptor stability and inhibiting constitutive downregulation. The modification of Ser residues in the receptor N-terminus by GalNAc-transferase 2 was affected by the SNP, which presents another distinction in the cellular processing of the two variants. The findings highlight the importance of the biosynthetic pathway in the regulation of GPCR behavior and pave way for strategies for treatments targeting GPCRs at this level. / Tiivistelmä Solujenvälisellä viestinnällä on keskeinen tehtävä kehon kaikissa toiminnoissa. δ-opioidireseptori (δOR) on solusignalointiin erikoistuneen kalvoproteiiniperheen (G-proteiiniin kytketyt reseptorit) jäsen, joka ohjaa kivuntuntemusta ja mielialoja. Sitä pidetään mahdollisena lääkekehityksen kohteena paitsi kivunlievityksen, myös useiden neurologisten häiriöiden hoidossa. δOR ilmenee kahtena polymorfisena muotona sen solunulkoisessa osassa tapahtuneen aminohappomuutoksen vuoksi (Phe27Cys). Työssä tutkittiin reseptorin glykosylaatiota ja dimerisaatiota, jotka säätelevät sen prosessointia, käyttäytymistä ja toimintaa. Käyttäen useita biokemiallisia ja solubiologisia menetelmiä työssä osoitettiin polymorfian vaikuttavan useisiin prosessointivaiheisiin ja muokkaavan siten reseptorin viestintää. Proteiinien laadunvalvontakoneiston havaittiin säätelevän reseptorin siirtymistä endoplasmakalvostolta solun pinnalle kahdella eri mekanismilla ohjaten osan reseptoreista hajotukseen. Toisin kuin Phe27-variantin, tehottomasti kypsyvän Cys27-variantin laadunvalvonta on riippuvainen reseptoriin liittyvistä N-glykaaneista ja näihin sitoutuvasta kaitsijaproteiinista, kalneksiinista. Reseptorivariantit, joista N-glykaanit puuttuvat, siirtyvät nopeammin solukalvolle, mutta ne ovat epästabiileja ja häviävät nopeasti solun pinnalta. Vaihtoehtoinen N-glykaaneista riippumaton laadunvalvontamekanismi sallii myös inaktiivisen Cys27-variantin pääsyn solun pinnalle. Varianttien dimerisoitumisen osoitettiin säätelevän niiden kuljetusta soluissa. Cys27-variantin havaittiin sitoutuvan Phe27-varianttiin aikaisessa biosynteesivaiheessa ja ohjaavan osan siitä hajotukseen. Tällä voi olla suuri merkitys opioidiviestinnässä molempia alleeleja kantavilla henkilöillä. Työssä havaittiin myös GalNAc-transferaasi-2-entsyymin ohjaavan Golgin laitteessa tapahtuvaa reseptorin O-glykosylaatiota. Se glykosyloi reseptorin solunulkoisen osan seriinitähteitä (Ser6, Ser25, Ser29), stabiloiden siten solun pinnan reseptoreita ja tehostaen niiden viestintää. Lisäksi havaittiin eroja varianttien O-glykosylaatiossa, mikä voi osaltaan selittää varianttien ilmentymisessä todettuja eroja. Tutkimus luo uutta tietoa biosynteesireitin merkityksestä G-proteiiniin kytkettyjen reseptorien säätelyssä sekä antaa pohjaa keinoille, joilla tätä voitaisiin hyödyntää farmakologisesti.