FK506-binding protein 11, a novel antibody folding catalyst in plasma cells

Antibodies are glycoproteins produced by terminally differentiated B cells known as plasma cells. They are the central effectors of adaptive immunity, as they specifically bind invading pathogens leading to their neutralization. Besides their substantial function in the immune system, they are used as monoclonal antibodies for the treatment of many diseases, including cancer and autoimmune disorders, and are therefore produced recombinantly in large numbers. In the form of autoantibodies, on the other hand, antibodies may represent disease causing and promoting molecules. All processes mediated by antibodies rely on a functional, three-dimensional structure, which in turn is attained in the endoplasmatic reticulum (ER) and aided by several ER-resident chaperones and folding catalysts. Therefore, a profound knowledge of antibody folding is substantial not only to gain a deeper understanding of the immune system, but also to improve recombinant antibody folding and to develop novel strategies to deal with autoimmune diseases. Still, antibody folding is insufficiently characterized so far. Here, expression, localization, regulation and function of FKBP11, a potential novel antibody folding peptidyl-prolyl cis-trans isomerase (PPIase), was analyzed. Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease (ILD) with increasing incidence worldwide and a poor prognosis with median survival rates of 3-5 years upon diagnosis. It is a fibrotic disease defined by an excessive deposition of extracellular matrix (ECM) resulting in an irreversible, ultimately fatal disruption of the lung architecture. Treatment with the Food and Drug Administration (FDA) approved antifibrotic compounds pirfenidone and nintedanib can slow down disease progression, but does not stop it, highlighting the need for a better understanding of this disease along with novel treatment strategies. Importantly, there is emerging evidence of autoimmune features present in IPF, including lymphocytic aggregates within IPF lung tissue and elevated levels of autoantibodies in the serum of IPF patients. A better understanding of these features could provide novel therapeutic targets in the treatment of IPF. Consistent with preceding proteomics data, protein levels of FKBP11 were highly increased in lung tissue of IPF patients. In IPF lungs, immunofluorescence revealed that FKBP11 was specifically expressed by CD27+/CD38+/CD138+/CD3-/CD20-/CD45- plasma cells, the number of which was drastically elevated in IPF lungs. Accordingly, in vitro B cell to plasma cell differentiation induced by a mixture of Interleukin-2 (IL-2) and R848 (resiquimod) was accompanied by an upregulation of FKBP11. More specifically, FKBP11 was upregulated as a part of the unfolded protein response (UPR) in an XBP1-dependent manner, with XBP1 being an important driver of the plasma cell differentiation process. This was assessed by artificial induction of ER stress using tunicamycin upon two different cell lines. Interestingly, prior knockdown of FKBP11 made the cells more susceptible to ER stress induced cell death. Finally, the function of FBKP11 was determined using an in vitro antibody folding assay, showing that addition of human recombinant FKBP11 increased both the speed of antibody folding as well as total yields of correctly folded antibodies. This effect was inhibited by prior incubation of FKBP11 with tacrolimus (FK506). In agreement with a function in antibody folding, knockdown of FKBP11 in an antibody secreting hybridoma cell line reduced antibody levels in the cell culture supernatant. Overall, FKBP11 was identified as a novel, plasma cell specific antibody folding catalyst in IPF. This provides new insights into plasma cell biology and the process of antibody folding, supporting a role of autoimmunity in IPF and allowing for the conception of innovative, targeted therapies not only in IPF, but also in autoimmune disorders. Moreover, these insights may help to overcome present limitations in the production of therapeutic, monoclonal antibodies.Antikörper sind Glykoproteine, die von ausdifferenzierten B-Zellen, den Plasmazellen, produziert werden. Sie sind zentraler Bestandteil des adaptiven Immunsystems, indem sie eingedrungene Erreger binden und somit zu deren Beseitigung führen. Außerdem werden sie als monoklonale Antikörper zur Behandlung vieler Krankheiten, wie etwa Krebs und Autoimmunerkrankungen, genutzt und daher in großen Mengen rekombinant hergestellt. Andererseits können Antikörper auch in Form von Autoantikörpern zur Entstehung von Krankheiten führen und diese aufrecht erhalten. All diese Prozesse, die durch die Bindung von Antikörpern ausgelöst werden, erfordern eine funktionierende, dreidimensionale Struktur des Antikörpers, die unter Zuhilfenahme von Chaperonen und Faltungskatalysatoren im endoplasmatischen Retikulum (ER) entsteht. Daher ist ein tiefgreifendes Verständnis von Antikörperfaltung nicht nur wesentlich, um neue Erkenntnisse zum Immunsystem zu gewinnen, sondern kann auch die rekombinante Herstellung monoklonaler Antikörper verbessern sowie dazu beitragen, neue Strategien zur Behandlung von Autoimmunerkrankungen zu gewinnen. Bislang ist der Vorgang der Antikörperfaltung jedoch unzureichend dargestellt worden. In dieser Arbeit wurde die Expression, Lokalisation, Regulation sowie Funktion von FKBP11, einer Peptidyl-Prolyl-cis/trans-Isomerase (PPIase), im Hinblick auf eine mögliche Funktion als antikörperfaltendes Enzym analysiert. Die idiopathische Lungenfibrose (IPF) gehört zu den interstitiellen Lungenerkrankungen (ILDs) und hat eine schlechte Prognose mit einer mittleren Überlebenszeit von 3-5 Jahren bei Diagnosestellung. Die Inzidenz der IPF ist weltweit steigend. Die Krankheit ist gekennzeichnet durch eine zunehmende Vernarbung (Fibrosierung) des Lungengewebes, die durch eine enorme Ablagerung von extrazellulärer Matrix (ECM) gekennzeichnet ist. Dies führt zu einer zunehmenden Zerstörung der Lungenarchitektur, die am Ende zum Tod der Patienten führt. Die derzeit von der Arzneimittelzulassungsbehörde der USA (Food and Drug Administration, FDA) zugelassenen, anti-fibrotischen Medikamente, namentlich Pirfenidon und Nintedanib, verlangsamen lediglich das Voranschreiten der Erkrankung, verhindern es jedoch nicht. Dies verdeutlicht, dass ein besseres Verständnis der IPF notwendig ist, um neue Behandlungsansätze zu identifizieren. Weiterhin wird zunehmend ersichtlich, dass Eigenschaften von Autoimmunerkrankungen in der IPF zu finden sind, wie etwa lymphozytäre Infiltrate in den Lungen der Patienten sowie zirkulierende Autoantikörper im Serum der Patienten. Eine bessere Charakterisierung dieser autoimmunen Eigenschaften in der IPF könnte dazu führen, neue Behandlungsansätze zu verwirklichen. Übereinstimmend mit einer vorhergehenden Proteomikstudie der IPF zeigten Lungengewebeproben von IPF-Patienten höhere Proteinmengen von FKBP11. In der Immunfluoreszenz war ersichtlich, dass FKBP11 in IPF Lungen ausschließlich von Plasmazellen (CD27+/CD38+/CD138+/CD3-/CD20-/CD45-) exprimiert wird. Entsprechend war die Zahl der Plasmazellen in IPF-Lungen deutlich erhöht. Damit übereinstimmend führte eine in vitro Plasmazelldifferenzierung mithilfe von Interleukin-2 (IL-2) und R848 (Resiquimod) zu einer Hochregulierung von FKBP11. Eine künstliche Herbeiführung von ER-Stress mittels Tunicamycin in zwei unabhängigen Zelllinien führte zur Hochregulierung der ungefalteten Protein-Antwort (UPR) und zeigte auf, dass FKBP11 als Teil der UPR in der Plasmazelldifferenzierung hochreguliert wird. Genauer noch bestätigte der vorhergehende Knockdown von XBP1, einem wichtigen Transkriptionsfaktor der Plasmazelldifferenzierung, dass die Hochregulierung von FKBP11 über XBP1 vermittelt wird. Weiterhin zeigte sich bei Herbeiführung von ER-Stress nach vorhergehendem Knockdown von FKBP11 eine erhöhte Empfindlichkeit der Zellen gegenüber ER-Stress-induziertem Zelltod. Letztlich konnte durch ein in vitro Antikörperfaltungssystem demonstriert werden, dass rekombinantes, humanes FKBP11 die Fähigkeit besitzt, Antikörper in vitro zu falten, da es sowohl den Faltungsprozess selbst beschleunigte, als auch den Ertrag an korrekt gefalteten Antikörpern steigerte. Diese Effekte konnten durch vorhergehende Inkubation von FKBP11 mit Tacrolimus (FK506) verhindert werden. Damit übereinstimmend führte ein Knockdown von FKBP11 in einer antikörperproduzierenden Hybridomzelllinie zu geringeren Antikörperkonzentrationen im Zellkulturüberstand. Abschließend lässt sich sagen, dass mit FKBP11 ein neuartiges Protein identifiziert wurde, das in der Lage ist, Antikörper in Plasmazellen zu falten. Dies gewährt neuartige Einblicke in die Biologie der Plasmazelle sowie den Prozess der Antikörperfaltung, und unterstützt weiterhin die Rolle von Autoimmunität in der IPF. Dies ermöglicht die Entwicklung neuartiger Therapieansätze, sowohl in der Behandlung der IPF, als auch in der Behandlung von Autoimmunerkrankungen. Darüber hinaus können diese Erkenntnisse dazu beitragen, die Herstellung rekombinanter, monoklonaler Antikörper zu verbessern

Loss of PRDM1/BLIMP-1 function contributes to poor prognosis of activated B-cell-like diffuse large B-cell lymphoma

PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novo DLBCL was effectively evaluated for PRDM1/BLIMP-1 deletion, mutation, and protein expression. BLIMP-1 expression was frequently associated with the ABC phenotype and plasmablastic morphologic subtype of DLBCL, yet 63% of the ABC-DLBCL patients were negative for BLIMP-1 protein expression. In these patients, loss of BLIMP-1 was associated with Myc overexpression and decreased expression of p53 pathway molecules. In addition, homozygous PRDM1 deletions and PRDM1 mutations within exons 1 and 2, which encode for domains crucial for transcriptional repression, were found to show a poor prognostic impact in patients with ABC-DLBCL but not in those with germinal center B-cell-like DLBCL (GCB-DLBCL). Gene expression profiling revealed that loss of PRDM1/BLIMP-1 expression correlated with a decreased plasma-cell differentiation signature and upregulation of genes involved in B-cell receptor signaling and tumor-cell proliferation. In conclusion, these results provide novel clinical and biological insight into the tumor-suppressive role of PRDM1/BLIMP-1 in ABC-DLBCL patients and suggest that loss of PRDM1/BLIMP-1 function contributes to the overall poor prognosis of ABC-DLBCL patients

FK506-binding proteins : identification and analysis in Neurospora crassa

Mestrado em MicrobiologiaImunofilinas são receptores intracelulares de drogas imunossupressoras, como a ciclosporina A, FK506 e rapamicina, caracterizados por apresentarem actividade de peptidil-prolil cis-trans isomerase. Além do seu envolvimento no enovelamento de proteínas, novos estudos demonstram o envolvimento destas proteínas em processos celulares específicos como transdução de sinal, transporte e montagem de complexos proteícos e silenciamento de rDNA. Neste estudo propusemo-nos caracterizar as funções celulares das imunofilinas do tipo FKBP (FK506-binding proteins) identificadas em Neurospora crassa. Na procura pelo papel fisiológico destas proteínas confirmamos a localização celular das proteínas FKBP12, FKBP22 e FKBP50 e determinamos a localização subcelular da proteína FKBP11. Foi também analisada a expressão destas proteínas em resposta a diversos indutores de stress, realizadas imunoprecipitações e produzidos e analisados mutantes simples e duplos para os genes fkbp. Através da proteína de fusão FKBP50::GFP e extractos proteicos da estirpe selvagem foi possível confirmar a localização nuclear da FKBP50, a localização endoplasmática da FKBP22 e a localização mitocondrial e citoplasmática da FKBP13. Foi também possível determinar que a FKBP11 está localizada no citoplasma. Uma análise bioquímica mais detalhada destas proteínas demonstrou que a FKBP11 não é expressa durante o desenvolvimento vegetativo do fungo mas a sua expressão é induzida por elevados níveis de cálcio e durante o desenvolvimento sexual. Através de imunoprecipitações usando um soro contra a FKBP13 foi possível co-imunoprecipitar uma proteína de aproximadamente 40 kDa. O tratamento de extractos proteícos mitocondriais e citoplasmáticos com diferentes proteases demonstrou que a FKBP13 é extremamente resistente à proteólise por proteinase K e tripsina. A análise da expressão destas proteínas em resposta a diferentes indutores de stress sugere que a FKBP50 é regulada negativamente pelo cálcio. Os níveis de FKBP22 encontram-se mais elevados em resposta ao stress osmótico e oxidativo e os níveis de FKBP13 encontramse também aumentados na presença de elevados níveis de FKBP11, sugerindo uma possível ligação entre FKBP13 e FKBP11. Embora a disrupção dos genes fkbp11, fkbp13 e fkbp22 não conduza a num fenótipo detectável, a disrupção do gene fkbp50 resulta num fenótipo termo-sensível. No seu conjunto, os resultados este estudo demonstram que além do envolvimento no enovelamento proteíco, estas proteínas apresentam um conjunto de características particulares, provavelmente como resultado de funções específicas. Com a caracterização das funções celulares realizadas por estas proteínas, esperamos descobrir um pouco mais sobre o papel das imunofilinas em processos celulares fundamentais comuns a todos os eucariotas. ABSTRACT: Immunophilins are intracellular receptors of immunosuppresive drugs, as cyclosporin, FK506 and rapamycin, found to possess peptidyl-prolyl cis-trans isomerase activity. In addition to their involvement in protein folding, new data demonstrate the participation of these proteins in specific cellular processes as signal transduction, protein trafficking and assembly and rDNA silencing. In this study we proposed to characterize the cellular functions of the immunophilins of the FKBP-type (FK506-binding proteins) identified in Neurospora crassa. In a search for the physiological role of these proteins we confirmed the cellular location of proteins FKBP13, FKBP22 and FKBP50 and determined the subcellular location of the FKBP11. We also analyzed the expression of these proteins in response to several stress inducers, performed immunoprecipitations and produced and analyzed single and multiple mutants for the fkbp genes. Using a GFP fusion protein and wild type protein extracts we confirmed the nuclear location of FKBP50, the endoplasmic location of FKBP22 and the mitochondrial and cytoplasmic location of FKBP13. We also determine that FKBP11 is located in cytoplasm. A more detailed biochemical analysis of these proteins revealed that FKBP11 is not expressed during vegetative development of the fungus but can be induced by high levels of calcium and during sexual development. Through immunoprecipitation using an antiserum against the FKBP13 we could coimmunoprecipitate a protein of approximately 40 kDa. Treatment of wild type mitochondrial and cytoplasmic protein extracts with different proteases showed that FKBP13 is highly resistant to proteolysis by proteinase K and trypsin. Phenotypic analysis of the FKBPs expression in response to several stress inducers revealed that FKBP50 might be down regulated by calcium. The FKBP22 levels were found to be specially elevated in response to osmotic and oxidative stress and FKBP13 levels can be found notably high in the presence of high amounts of FKBP11, suggesting a possible relation between FKBP13 and FKBP11. Although disruption of the fkbp11, fkbp13 and fkbp22 genes did not conducted to a detectable phenotype, disruption of the fkbp50 gene resulted in a temperature-sensitive phenotype. All together the results of this work showed that besides the involvement in protein folding these proteins present particular features, probably as a result of specific cellular functions. With the characterization of the cellular functions mediated by these proteins we expect to find out a little more about the role of immunophilins in fundamental cellular processes common to all eukaryotes

Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

Abstract Background Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response. Results Expression of Ins 2 (C96Y)-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6) and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3) following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp protein expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival. Conclusions The inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in β-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes

Angiotensin II causes b-cell dysfunction through an ER stress-induced proinflammatory response

The metabolic syndrome is associated with an increase in the activation of the renin angiotensin system, whose inhibition reduces the incidence of new-onset diabetes. Importantly, angiotensin II (AngII), independently of its vasoconstrictor action, causes b-cell inflammation and dysfunction, which may be an early step in the development of type 2 diabetes. The aim of this study was to determine how AngII causes b-cell dysfunction. Islets of Langerhans were isolated from C57BL/6J mice that had been infused with AngII in the presence or absence of taurineconjugated ursodeoxycholic acid (TUDCA) and effects on endoplasmic reticulum (ER) stress, inflammation, and b-cell function determined. The mechanism of action of AngII was further investigated using isolated murine islets and clonal b cells. We show that AngII triggers ER stress, an increase in the messenger RNA expression of proinflammatory cytokines, and promotes b-cell dysfunction in murine islets of Langerhans both in vivo and ex vivo. These effects were significantly attenuated by TUDCA, an inhibitor of ER stress. We also show that AngII-induced ER stress is required for the increased expression of proinflammatory cytokines and is caused by reactive oxygen species and IP3 receptor activation. These data reveal that the induction of ER stress is critical for AngII-induced b-cell dysfunction and indicates how therapies that promote ER homeostasis may be beneficial in the prevention of type 2 diabetes. © 2017 Endocrine Society

A reevaluation of the role of the unfolded protein response in islet dysfunction: maladaptation or a failure to adapt?

Endoplasmic reticulum (ER) stress caused by perturbations in ER homeostasis activates an adaptive response termed the unfolded protein response (UPR) whose function is to resolve ER stress. If unsuccessful, the UPR initiates a proapoptotic program to eliminate the malfunctioning cells from the organism. It is the activation of this proapoptotic UPR in pancreatic β-cells that has been implicated in the onset of type 2 diabetes and thus, in this context, is considered a maladaptive response. However, there is growing evidence that β-cell death in type 2 diabetes may not be caused by a maladaptive UPR but by the inhibition of the adaptive UPR. In this review, we discuss the evidence for a role of the UPR in β-cell dysfunction and death in the development of type 2 diabetes and ask the following question: Is β-cell dysfunction the result of a maladaptive UPR or a failure of the UPR to adequately adapt? The answer to this question is critically important in defining potential therapeutic strategies for the treatment and prevention of type 2 diabetes. In addition, we discuss the potential role of the adaptive UPR in staving off type 2 diabetes by enhancing β-cell mass and function in response to insulin resistance

The human FK506-binding proteins: characterization of human FKBP19

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas

Androgen Regulated Genes in Human Prostate Xenografts in Mice: Relation to BPH and Prostate Cancer

Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues