A molecular specificity code for the three mammalian KDEL receptors

AC-terminal KDEL-like motif prevents secretion of soluble endoplasmic reticulum (ER)–resident proteins. This motif interacts with KDEL receptors localized in the intermediate compartment and Golgi apparatus. Such binding triggers retrieval back to the ER via a coat protein I–dependent pathway. To date, two human KDEL receptors have been reported. Here, we report the Golgi localization of a third human KDEL receptor. Using a reporter construct system from a screen of 152 variants, we identified 35 KDEL-like variants that result in efficient ER localization but do not match the current Prosite motif for ER localization ([KRHQSA]-[DENQ]-E-L). We cloned 16 human proteins with one of these motifs and all were found in the ER. A subsequent screen by bimolecular fluorescence complementation determined the specificities of the three human KDEL receptors. Each KDEL receptor has a unique pattern of motifs with which it interacts. This suggests a specificity in the retrieval of human proteins that contain different KDEL variants

Introduction of a More Glutaredoxin-like Active Site to PDI Results in Competition between Protein Substrate and Glutathione Binding

Proteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for PDI also in vivo, but at least for oxidation reactions, GSSG probably is not the major electron acceptor and PDI may not have evolved to react with glutathione with high affinity, but merely having adequate affinity for both glutathione and folding proteins/peptides. Glutaredoxins, on the other hand, have a high affinity for glutathione. They commonly have -CXFC- or -CXYC- active site, where the tyrosine residue forms part of the GSH binding groove. Mutating the active site of PDI to a more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the active site, as well as a decreased rate of the native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. This suggests that these variants lead to competition in binding between glutathione and folding protein substrates

Introduction of a More Glutaredoxin-like Active Site to PDI Results in Competition between Protein Substrate and Glutathione Binding

Proteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for PDI also in vivo, but at least for oxidation reactions, GSSG probably is not the major electron acceptor and PDI may not have evolved to react with glutathione with high affinity, but merely having adequate affinity for both glutathione and folding proteins/peptides. Glutaredoxins, on the other hand, have a high affinity for glutathione. They commonly have -CXFC- or -CXYC- active site, where the tyrosine residue forms part of the GSH binding groove. Mutating the active site of PDI to a more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the active site, as well as a decreased rate of the native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. This suggests that these variants lead to competition in binding between glutathione and folding protein substrates

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA) : characterisation of a novel cerebropulmonary disease

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.Peer reviewe

Henkilöstöhallinnon esimiesopas : Case: Hyvinkään kaupunki

Opinnäytetyöni tarkoituksena oli laatia Hyvinkään kaupungin palveluksessa toimivien esimiesten käyttöön esimiesopas, joka helpottaa heidän joka päivästä työskentelyään henkilöstöhallintoon liittyvien asioiden hoidossa, yhtenäistää asioiden hoitoon liittyviä käytäntöjä ja toimintatapoja sekä helpottaa kaupungin henkilöstökeskuksen työn kuormitusta. Opinnäytetyössäni on kaksi osaa. Teoriaosuudessa käsitellään toimintaympäristöä, johon esimiesoppaani kuuluu ja etenkin Hyvinkään kaupunkia työnantajana. Sen jälkeen tarkastelen julkisyhteisöjen palvelussuhdepiirteitä ja niihin liittyviä työ- ja virkamiesoikeudellisia työnantajavelvoitteita, joista esimiesten on julkisyhteisöjen edustajina huolehdittava. Lisäksi käsittelen kunta-alan työ- ja virkaehtosopimusjärjestelmää, pääsopijajärjestöjä ja paikallista sopimista. Opinnäyteyöni toiminnallinen osuus on Hyvinkään kaupungille laadittu henkilöstöhallinnon esimiesopas, jossa käyn läpi esimiehen työssä tarpeellisia työ- ja virkamiesoikeudellisia asioita. Esimiesopas toteutettiin yhteistyössä henkilöstökeskuksen kanssa.Human resource management guide for superiors City of Hyvinkää The aim of my thesis was to create a guide for the superiors who work for the city of Hyvinkää. The goal is to assist their everyday work in issues that relate to human resources management, unify, procedures that are used and facilitate the workload given to the people who work in the human resource management. My thesis consist of two parts. In the theory section the focus is in working environment where this guide is meant for. Also the section tells you more about the city of Hyvinkää as an employer. After that I look at the features of a public corporation as an employer and employer obligations between employee and employer, obligations the superiors must deal with as representatives of a public corporation. In addition, I clarify the structure of municipal collective service and labor agreements and I look at some of the top contracting organizations and local agreements. The functional part of my thesis is the human resource management guide for superiors. I created it for superiors who work for the city of Hyvinkää. The guide that I have compiled contains necessary legal facts that are related to working and service agreements and issues whom superior face in their daily work

Toista lastaan odottavien perheiden tuen tarve

Opinnäytetyön tarkoituksena oli kartoittaa toista lastaan odottavien perheiden tuen tarvetta. Tutkimuksemme oli kvantitatiivinen eli määrällinen tutkimus. Aineisto kerättiin opinnäytetyömme tutkimusta varten tehdyllä kyselylomakkeella, joka oli puolistrukturoitu. Kyselylomakkeessa oli sekä monivalinta- että avoimia kysymyksiä. Kyselylomakkeet jaettiin Porin seudun äitiysneuvoloihin ja terveydenhoitajat jakoivat kyselylomakkeet toista lastaan odottaville vanhemmille. Kyselylomakkeita jaettiin 184 kappaletta, joista palautui 69. Tutkimuksen vastausprosentti oli 37,5 %. Vastaukset analysoitiin Excelin Tixel- taulukkolaskentaohjelmalla. Tulokset esitettiin prosentteina, kuvioina ja taulukkoina. Avointen kysymysten vastaukset esitettiin suorina lainauksina. Tutkimuksen tulosten mukaan toinen raskaus muutti vastaajien perhe-elämää, parisuhdetta tai esikoisen käyttäytymistä jonkin verran. Muutoksia, joita perhe-elämässä tapahtui, olivat isän lisääntynyt osallistuminen esikoisen ja kodin hoitoon. Toinen raskaus vaikutti parisuhteeseen vähentämällä seksin määrää, lisäämällä ristiriitoja tai lähentämällä vanhempia. Vastauksista ilmeni, että jotkut esikoiset odottivat tulevaa vauvaa, toiset taas olivat huomionhakuisia, mustasukkaisia ja kiukuttelevia. Tuloksista ilmeni, että isän ja esikoisen huomioiminen äitiysneuvolassa koettiin tärkeäksi. Vanhemmat tarvitsivat terveydenhoitajalta aikaa tiedon jakamiseen, keskusteluun, kuunteluun ja läsnäoloon. Tietoa ja ohjausta he tarvitsivat raskausajasta, raskauden aikaisista häiriöistä, omasta hyvinvoinnista, esikoisen tukemisesta elämänmuutoksessa ja synnytyksestä. Vanhemmat tarvitsivat yksilöllistä, moniammatillista ja perhekeskeistä tukea. Vanhemmista suurin osa tarvitsi tukea toisen raskauden aikana. Tulosten mukaan terveydenhoitaja käytti vanhempien kanssa eniten aikaa tiedon jakamiseen, keskusteluun, kuunteluun ja läsnäoloon, joihin he aikaa tarvitsivatkin. Äidit saivat perheenjäsenistä eniten tukea. Vanhemmat saivat tietoa ja ohjausta raskausajasta, raskauden aikaisista häiriöistä ja omasta hyvinvoinnista. Vastaajista noin puolet koki saaneensa yksilöllistä tukea ja noin kolmasosa moniammatillista ja perhekeskeistä tukea. Vanhemmat saivat tukea melko hyvin. Tuloksista voi päätellä, että vanhempien tuen tarve ja saanti eivät aina vastanneet toisiaan. Tutkimustulostemme pohjalta terveydenhoitajat voivat kehittää toimintaansa vastaamaan paremmin toista lastaan odottavien perheiden tarpeisiin.The purpose of this thesis was to survey the need of support for families who are expecting a second child. The study method is quantitative. Material was collected with a semi-structured questionnaire made for the thesis and it consisted of both multiple choice questions and open-ended questions. The questionnaires were distributed to maternity clinics in Pori region. Nurses handed out the questionnaires to parents who were expecting a second child. A total number of 184 questionnaires were distributed, of which 69 were returned. The response rate was 37,5 %. The answers were analysed with Excel’s Tixel spreadsheet. The results were demonstrated in percentages, charts and tables. The answers of open-ended questions were expressed in direct quotations. According to the study’s results, the second pregnancy changed the respondents’ family lives, relationships, and the behaviour of the first child to a certain extent. Changes that took place in the families were fathers’ increased participation in care of home and the first born child. The second pregnancy affected relationships by decreasing the amount of sex, increasing conflicts or bringing the parents closer to each other. The responses showed that while some first born children were looking forward to the new baby, others were increasingly attention-seeking, jealous and capricious. The results mainly showed that paying attention to the father and the first child were considered important. Parents needed time from the nurse for providing information, conversation, listening and being present. They needed information and guidance about gestation and gestational disorders, own well-being, supporting the first child, and about birth. The parents needed individualized, professionally diversified and family-oriented support. The majority of parents needed support during the second pregnancy. According to the results, the nurse used most of the time with parents in the kind of support which they needed. The mothers received the most support among the family members. The parents got information and guidance about the gestation time, gestational disorders and own well-being. Approximately half of the respondents felt they got individualized support, while about a third got professionally diversified and family-oriented support. The results conclude that the support given to the parents did not correspond all the time to the kind of support which they needed. On the basis of our study results nurses can develop their activities to better match the needs of families who are expecting their second child

Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity

Abstract Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different enzymes. We engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their activity using a homogeneous NAD⁺ conversion assay, facilitating its automation to lower the assay volume and costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity

Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member

Native disulfide bond formation in the endoplasmic reticulum is a critical process in the maturation of many secreted and outer membrane proteins. Although a large number of proteins have been implicated in this process, it is clear that our current understanding is far from complete. Here we describe the functional characterization of a new 18-kDa protein (ERp18) related to protein-disulfide isomerase. We show that ERp18 is located in the endoplasmic reticulum and that it contains a single catalytic domain with an unusual CGAC active site motif and a probable insertion between beta3 and alpha3 of the thioredoxin fold. From circular dichroism and NMR measurements, ERp18 is well structured and undergoes only a minor conformational change upon dithioldisulfide exchange in the active site. Guanidinium chloride denaturation curves indicate that the reduced form of the protein is more stable than the oxidized form, suggesting that it is involved in disulfide bond formation. Furthermore, in vitro ERp18 possesses significant peptide thiol-disulfide oxidase activity, which is dependent on the presence of both active site cysteine residues. This activity differs from that of the human PDI family in that under standard assay conditions it is limited by substrate oxidation and not by enzyme reoxidation. A putative physiological role for Erp18 in native disulfide bond formation is discussed