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Physiological Role of Prolylcarboxypeptidase

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Physiological Role of Prolylcarboxypeptidase Physiological Role of Prolylcarboxypeptidase Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium ( D r. r e r. n a t .) eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I Humboldt-Universität zu Berlin von Dipl. Biologin Ines Claudia Schadock Präsident der Humboldt-Universität zu Berlin: Prof. Dr. Jan-Hendrik Olbertz Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I : Prof. Dr. Andreas Herrmann Gutachter/in: 1. Prof. Dr. rer. nat. Michael Bader 2. Prof. Dr. rer. nat. Nobert Hübner 3. Prof. Dr. rer. nat. Achim Leutz Datum der mündlichen Prüfung: 20.06.2011 ABSTRACT Prolylcarboxypeptidase (PRCP, EC3.4.16.2) is an enzyme specifically cleaving the last carboxy-terminal amino acid from substrates containing a penultimate proline. Its known potential substrates are linked to cardiovascular and metabolic phenomenon. To analyse the in vivo function of this enzyme a PRCP knockout mouse was gener- ated. Homozygous knockout mice are viable but show tendency of decreased life span. In mice prcp expression is present in all tissues tested with very specific local- izations of prcp promotor activity to distinct brain areas within the cortex, hippocam- pus, hypothalamus and the brain stem. The metabolic phenotype of PRCP deficient mice is characterized by low body weight even when feeding the animals a high fat diet. The increased plasma leptin levels and elevated expression of proopiomelanocortin gene (pomc) found in knockout hy- pothalami suggests an involvement of PRCP in the regulation of food intake and en- ergy homeostasis. One of the gene products of pomc is α-melanocortin stimulating hormone that is terminating feeding when released from hypothalamic POMC neu- rons. Its carboxy-terminal structure is fitting the cleavage preferences of PRCP. Prcp promotor activities are localized in arcuate nucleus and paraventricular nucleus, brain areas of known αMSH signalling, supporting a role of PRCP in the degradation of central αMSH. The impact of PRCP on angiotensin II (AngII) metabolism was studied by determin- ing the level of AngII and its degradation product Ang1-7 in blood and tissues. But instead of increased AngII levels due to the missing degradation enzyme in knockout mice, the breakdown product Ang1-7 was found increased in kidney and white adi- pose tissue. These results were explainable by the increased activity of angiotensin converting enzyme 2 (ACE2) found in kidney. Probably ACE2 is compensating the lack of PRCP in the knockout mouse. Nevertheless, blood pressure and heart rate of PRCP knockout mice was increased. The mild hypertension was accompanied by mild hypertrophy of the hearts. Prcp promotor activity was found in brain stem areas im- I portant for regulation of blood pressure and heart rate suggesting that central PRCP regulates blood pressure. Keywords: prolylcarboxypeptidase, angiotensin II, alpha-melanocortin stimulating hor- mone, hypertension, obesity II ZUSAMMENFASSUNG Die Namen-gebende Hauptcharakteristik von Prolylcarboxypeptidase (PRCP, EC3.4.16.2) ist spezifisch die letzte carboxyterminale Aminosäure von Substraten abzuspalten, deren vorletzte Aminosäure ein Prolin ist. Seine bisher publizierten Sub- strate Angiotensin II (AngII) und α-Melanocortin Stimulierendes Hormone (αMSH) legen eine Rolle von PRCP in der Entwicklung von kardiovaskulären und metaboli- schen Krankheiten nahe. Um die in vivo Funktion von PRCP zu studieren, wurde eine Knockout Maus generiert. Die homozygoten Tiere dieser Linie zeigten von Geburt an keine offensichtlichen Beeinträchtigungen. Allerdings beschreibt eine Langzeitstudie der Tiere eine Tendenz verkürzter Lebenserwartung im Vergleich zu Kontrolltieren. Die Expression von prcp in der Maus ist in allen getesteten Organen vorhanden, wo- bei die höchste Expression im Gehirn gefunden wurde. Hier wurde zudem prcp Pro- motoraktivität in eng begrenzten Bereichen von Cortex, Hippocampus, Hypothalamus und Hirnstamm lokalisiert. PRCP Knockoutmäuse wiesen generell ein reduziertes Körpergewicht auf, selbst wenn sie über Monate mit einer Hochfettdiät versorgt wurden. Erhöhte Plasmaleptin Werte und Proopiomelanocortin (pomc) Expression in knockout Hypothalami wiesen auf eine wichtige Rolle von PRCP in der Regulation von Futteraufnahme und Energieho- möostase hin. Eines der Genprodukte von pomc ist αMSH, welches bei Freisetzung im Hypothalamus die Futteraufnahme terminiert. Die carboxyterminale Struktur die- ses Neuropeptids erfüllt alle Voraussetzungen, um von PRCP gespalten zu werden. Zudem konnte prcp Promotoraktivität in den selben Hirnstrukturen gezeigt werden, in denen auch αMSH-Wirkung beschrieben wurde. Eine mögliche Funktion von PRCP wäre somit die Inaktivierung des Appetitzüglers αMSH im Hypothalamus. Der Einfluß von PRCP auf den AngII Metabolismus, sollte durch Analyse von Peptid- konzentrationen ermittelt werden. Dabei stellte sich heraus, daß AngII Werte in PRCP Knockoutmäusen unverändert waren. Hingegen konnten paradoxerweise erhöhte Niveaus des Degradationsproduktes Ang1-7 in Niere und weißem Fettgewebe gezeigt werden. Die Entdeckung einer erhöhten Enzymaktivität von Angiotensin Converting III Enzyme 2 (ACE2) in Knockout Nieren, bot einen Erklärungsansatz für diese Ergebnis- se. Es wird davon ausgegangen, daß ACE2 die fehlende PRCP Aktivität in knockout Mäusen kompensiert. Trotzdem sind Blutdruck und Herzrate in PRCP Knockoutmäu- sen erhöht. Die milde Hypertension resultiert zusätzlich in einer leichten Herzhyper- trophie. Da die spezifische prcp Promotoraktivität in Hirnnuclei gefunden wurde, die in die Kontrolle der Herzfrequenz und des Blutdrucks involviert sind, wird eine regula- torische Funktion von Hirnstamm-PRCP auf Herzrhytmus und Blutdruck vermutet. Schlagwörter: Prolylcarboxypeptidase, Angiotensin II, alpha-Melanocortin Stimmulierendes Hormon, Hypertension, Obesitas IV ACKNOWLEDGEMENT Mein besonderer Dank gilt Prof. Michael Bader für die Möglichkeit, dieses Thema zu bearbeiten, die stets ausgezeichnete Betreuung meiner Arbeit und für die erstklassi- gen Arbeitsbedingungen in seinem Labor. Dank seiner konstruktiven Kritik und Anre- gung war die Anfertigung dieser Arbeit erst möglich. Für die Möglichkeit, mein Methodenspektrum zu erweitern und etablierte Versuchs- anordnungen nutzen zu können, möchte ich mich bei Prof. R.A.S. Santos (Peptidmes- sungen, UFMG, Belo Horizonte, Brasilien), Dr. W.E. Siems (ACE2 und NEP Enzymakti- vitätsbestimmungen, FMP, Berlin), Prof. G. Lewin (Aktivitätsmessung von Mäusen, MDC, Berlin) sowie Dr. Arndt Heuser und Martin Taube (Echokardiographie und Klein- tier Körperkompositions Scan, MDC, Berlin) bedanken. Ein großes Dankeschön gilt meinen Arbeitskollegen der AG Bader. Namentlich erwäh- nen möchte ich Sabine Grüger und Manfred Ströhmann für die tatkräftige Unterstüt- zung im Tierhaus; Andrea Müller, Susanne de Costa Goncalves, Anne Köhn und Sonja Kumsteller, die den Laboralltag so reibungslos organisieren, dass man jederzeit gern dort arbeitet; Natalia Alenina, Fatimunissa Qadri (alias Sayeeda), Mihail Todiras, Ralf Plehm und Irina Lapidous, die mir mit Rat und Tat zu wissenschaftlichen Methodiken den Rücken gestärkt haben. Bei meiner Familie und bei meinen Freunden möchte ich mich für das große Ver- ständnis und die immerwährende Unterstützung bedanken, auch wenn sie viel auf meine Person verzichten mußten. V VI CONTENT ABSTRACT..............................................................................................................................I ZUSAMMENFASSUNG..........................................................................................................III ACKNOWLEDGEMENT ........................................................................................................... V CONTENT ........................................................................................................................... VII LIST OF FIGURES.................................................................................................................IX LIST OF TABLES .................................................................................................................... X 1 INTRODUCTION............................................................................................................ 1 1.1 What is Prolylcarboxypeptidase? ................................................................................... 1 1.2 Hormonal Control of Food Intake .................................................................................. 5 1.3 Renin Angiotensin System ............................................................................................ 9 2 AIMS OF THE STUDY ................................................................................................... 13 3 MATERIAL & METHODS............................................................................................... 15 3.1 Chemicals ................................................................................................................. 15 3.2 Kits, Enzymes and Markers ......................................................................................... 17 3.3 Lab Instruments, Machines and other Material ............................................................. 18 3.4 Antibodies................................................................................................................. 19 3.5 Oligonucleotides ........................................................................................................ 20 3.6 Molecular Biological Protocols ....................................................................................
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