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Platelet activation and aggregation at sites of vascular injury is critical to prevent excessive blood loss, but may also lead to life-threatening ischemic diseases, such as myocardial infarction and stroke. Extracellular agonists induce platelet activation by stimulation of platelet membrane receptors. Signal transduction results in reorganization of the cytoskeleton, shape change, platelet adhesion and aggregation, cumulating in thrombus formation. Several Rho GTPases, including Rac1, Cdc42 and RhoA, are essential mediators of subsequent intracellular transduction of ITAM- and GPCR-signaling. Therefore, inhibition or knockout can result in severely defective platelet signaling.
Mice with platelet specific Rac1-deficiency are protected from arterial thrombosis. This benefit highlights further investigation of Rac1-specific functions and its potential as a new pharmacological target for prevention of cardiovascular diseases. Two newly developed synthetic compounds, NSC23766 and EHT1864, were proposed to provide highly specific inhibition of Rac1 activity, but both drugs have never been tested in Rac1-deficient cell systems to rule out potential Rac1-independent effects.
This study revealed significant off-target effects of NSC23766 and EHT1864 that occurred in a dose-dependent fashion in both wild-type and Rac1-deficient platelets. Both inhibitors individually affected resting platelets after treatment, either by altering membrane protein expression (NSC23766) or by a marked decrease of platelet viability (EHT1864). Platelet apoptosis could be confirmed by enhanced levels of phosphatidylserine exposure and decreased mitochondrial membrane potential. Phosphorylation studies of the major effector proteins of Rac1 revealed that NSC23766 and EHT1864 abolish PAK1/PAK2 activation independently of Rac1 in wild-type and knockout platelets, which may contribute to the observed off-target effects.
Additionally, this study demonstrated the involvement of Rac1 in G protein-coupled receptor-mediated platelet activation and GPIb-induced signaling. Furthermore, the data revealed that Rac1 is dispensable in the process of integrin IIb 3-mediated clot retraction.
This study unveiled that new pharmacological approaches in antithrombotic therapy with Rac1 as molecular target have to be designed carefully in order to obtain high specificity and minimize potential off-target effects.
Phosphatasen der HAD (haloacid dehalogenase)-Familie sind weit verbreitet in allen Domänen des Lebens und erfüllen die verschiedensten zellulären Aufgaben, beispielsweise in Metabolismus und Zellregulation. Die HAD-Phosphatase Chronophin zeigt Phosphataseaktivität unter anderem gegenüber Pyridoxal-5‘-Phosphat (PLP), einem essentiellen Kofaktor vieler biochemischer Prozesse, und Phosphocofilin, einem Regulator des Aktinzytoskeletts. Chronophin dimerisiert über die Interaktion zweier identischer Untereinheiten zu einem Homodimer. Ziel dieser Arbeit war, die Rolle dieser Dimerisierung, eines bei HAD-Phosphatasen weit verbreiteten Oligomerisierungszustandes, näher zu untersuchen.
Hierzu wurde die Dimerisierung erfolgreich durch den Austausch der Aminosäuren Alanin 194 und 195 zu Lysinen (Mutation A194K/A195K) gestört. Der Nachweis einer konstitutiv monomeren Chronophin-Mutante mittels Größenausschlusschromatographie, Rasterkraftmikroskopie, analytischer Ultra¬zentrifugation und Zellexperimenten wurde schließlich über die Struktur¬auflösung mittels Röntgenstrukturanalyse bestätigt. Aktivitätsmessungen der monomeren Mutante gegenüber dem Substrat PLP zeigten eine deutliche Verminderung der Phosphataseaktivität. Die Röntgenstrukturanalyse von Chronophin A194K/A195K im Vergleich mit Wildtyp-Chronophin enthüllte einen Mechanismus, wie die sogenannte Substratspezifitätsschleife, die für die korrekte Positionierung des PLP sorgt, im Homodimer des Wildtyps durch Interaktionen mit dem zweiten Protomer stabilisiert wird. Diese Stabilisierung fehlt bei der monomeren Mutante und äußert sich in einer veränderten Stellung der Substratspezifitätsschliefe. Der Strukturvergleich von Chronophin mit weiteren HAD-Phosphatasen der selben strukturellen Untergruppe vom C2a-Typ lässt eine allgemeine Gültigkeit der hier beschriebenen allosterischen Kontrolle von Substratspezifität über Homodimerisierung bei HAD-Phosphatasen vermuten und könnte so neue Ansatzpunkte für möglicherweise auch therapeutisch nutzbare Aktivitätshemmungen liefern.
Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity
(2019)
Adaptation to alterations in nutrient availability ensures the survival of organisms. In vertebrates, adipocytes play a decisive role in this process due to their ability to store large amounts of excess nutrients and release them in times of food deprivation. In todays western world, a rather unlimited excess of nutrients leads to high-caloric food consumption in humans. Nutrient overload together with a decreased energy dissipation result in obesity as well as associated diseases such as insulin resistance, diabetes, and liver steatosis. Obesity causes a hormonal imbalance, which in combination with altered nutrient levels can aberrantly activate G-protein coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D (PKD) 1 is a DAG effector integrating multiple hormonal and nutritional inputs. Nevertheless, its physiological role in adipocytes has not been investigated so far. In this thesis, evidence is provided that the deletion of PKD1 in adipocytes suppresses lipogenesis as well as the accumulation of triglycerides. Furthermore, PKD1 depletion results in increased mitochondrial biogenesis as well as decoupling activity. Moreover, PKD1 deletion promotes the expression of the β3-adrenergic receptor (ADRB3) in a CCAAT/enhancer-binding protein (C/EBP)-α and δ-dependent manner. This results in elevated expression levels of beige markers in adipocytes in the presence of a β-agonist. Contrarily, adipocytes expressing a constitutive active form of PKD1 present a reversed phenotype. Additionally, PKD1 regulates adipocyte metabolism in an AMP-activated protein kinase (AMPK)-dependent manner by suppressing its activity through phosphorylation of AMPK α1/α2 subunits. Thus, PKD1 deletion results in an enhanced activity of the AMPK complex. Consistent with the in vitro findings, mice lacking PKD1 in adipocytes demonstrate a resistance to high-fat diet-induced obesity due to an elevated energy expenditure caused by trans-differentiation of white into beige adipocytes. Moreover, deletion of PKD1 in murine adipocytes improves systemic insulin sensitivity and ameliorates liver steatosis. Finally, PKD1 levels positively correlate with HOMA-IR as well as insulin levels in human subjects. Furthermore, inhibition of PKD1 in human adipocytes leads to metabolic alterations, which are comparable to the alterations seen in their murine counterparts. Taken together, these data demonstrate that PKD1 suppresses energy dissipation, drives lipogenesis, and adiposity. Therefore, increased energy dissipation induced by several complementary mechanisms upon PKD1 deletion might represent an attractive strategy to treat obesity and its related complications.
Die Phosphoglykolat-Phosphatase PGP (früher auch als AUM bezeichnet) wurde in unserem Labor als Mitglied der HAD-Typ-Phosphatasen identifiziert. Die genetische Inaktivierung des Enzyms im gesamten Mausorganismus führt ab E8.5 zu einer Wachstumsverzögerung muriner Embryonen und bis E12.5 schließlich zu deren Tod. Im Gegensatz dazu sind Mäuse mit einer PGP-Inaktivierung in hämatopoetischen Zellen und im Endothel lebensfähig und phänotypisch unauffällig. Neue Erkenntnisse schreiben dem Enzym neben einer Aktivität gegenüber Phosphoglykolat auch Aktivitäten gegenüber Glycerin-3-phosphat (G3P), P-Erythronat und P-Lactat zu. Da diese Phosphatase-Aktivitäten Auswirkungen auf den Lipidstoffwechsel nahelegen, wurde in der vorliegenden Arbeit mittels massenspektrometrischer Methoden der Einfluss der Phosphoglykolat-Phosphatase auf den Metabolismus von Signal-, Membran- und Speicherlipiden in murinen Embryonen und Lymphozyten untersucht.
Nach Inaktivierung der PGP im gesamten Organismus wurden in E8.5-Embryonen erhöhte Diacylglycerin (DG)-, Triacylglycerin (TG)- und Sphingomyelin (SM)-Spiegel gemessen, während niedrigere Phosphatidylcholin (PC)-Level vorlagen.
In PGP-inaktivierten Lymphozyten waren G3P-, DG-, TG-, PC- und SM-Level nicht verändert. Dafür kam es zu signifikanten Erhöhungen der Phosphatidylglycerol (PG*)- und Cardiolipin (CL)-Spiegel.
Zusammenfassend konnte gezeigt werden, dass die PGP in unterschiedlichen Geweben differenzielle Effekte auf die Spiegel verschiedener Lipide hat. Dies deckt neue Funktionen der PGP für die Regulation des Lipidmetabolismus auf. Die vorliegende Arbeit stellt somit die Grundlage für weitere Untersuchungen über die genauen Ursachen und Folgen dieser Regulation dar und lässt auf eine wichtige Rolle der PGP als metabolische Phosphatase im Organismus schließen.
Platelet activation and aggregation at sites of vascular injury involves massive cytoskeletal re-organization, which is required for proper platelet function. Moreover, the cytoskeleton plays central roles in megakaryo- and thrombopoiesis. Thus, cytoskeletal protein aberrations can be the underlying reason for many pathological phenotypes. Although intensive research is carried out to identify the key players involved in cytoskeletal reorganization, the signaling cascades orchestrating these complex processes are still poorly understood. This thesis investigates the role of three actin-binding proteins, Coactosin-like (Cotl) 1, Profilin (Pfn) 1 and Thymosin (T) β4, in platelet formation and function using genetically modified mice.
ADF-H-containing proteins such as Twinfilin or Cofilin are well characterized as regulators of thrombopoesis and cytoskeletal reorganization. Although Cotl1 belongs to the ADF-H protein family, lack of Cotl1 did not affect platelet count or cytoskeletal dynamics. However, Cotl1-deficiency resulted in significant protection from arterial thrombus formation and ischemic stroke in vivo. Defective GPIb-vWF interactions and altered second wave mediator release present potential reasons for the beneficial effect of Cotl1-deficiency. These results reveal an unexpected function of Cotl1 as a regulator of thrombosis and hemostasis, establishing it as a potential target for a safe therapeutic therapy to prevent arterial thrombosis or ischemic stroke.
Recent studies showed that the organization of the circumferential actin cytoskeleton modulates calpain-mediated αIIbβ3 integrin closure, thereby also controlling αIIbβ3 integrin localization. The second part of this thesis identified the actin-sequestering protein Pfn1 as a central regulator of platelet integrin function as Pfn1-deficient platelets displayed almost abolished αIIbβ3 integrin signaling. This translated into a profound protection from arterial thrombus formation and prolonged tail bleeding times in vivo which was caused by enhanced calpain-dependent integrin closure. These findings further emphasize the importance of a functional actin cytoskeleton for intact platelet function in vitro and in vivo.
Tβ4 is a moonlighting protein, acting as one of the major actin-sequestering proteins in cells of higher eukaryotes and exerting various paracrine functions including anti-inflammatory, immunomodulatory and pro-angiogenic effects. Although excessively studied, its role for cytoskeletal dynamics, the distinction between endo- and exogenous protein function and its uptake and release mechanisms are still poorly understood. Constitutive Tβ4-deficiency resulted in thrombocytopenia accompanied by a largely diminished G-actin pool in platelets and divergent effects on platelet reactivity. Pre-incubation of platelets with recombinant Tβ4 will help to understand the function of endo- and exogenous protein, which is under current investigation.
Platelets are continuously produced from megakaryocytes (MK) in the bone marrow by a cytoskeleton-driven process of which the molecular regulation is not fully understood.
As revealed in this thesis, MK/ platelet-specific Profilin1 (Pfn1) deficiency results in micro- thrombocytopenia, a hallmark of the Wiskott-Aldrich syndrome (WAS) in humans, due to accelerated platelet turnover and premature platelet release into the bone marrow. Both Pfn1-deficient mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyper-stable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients.
In contrast, Twinfilin2a (Twf2a) was established as a central regulator of platelet reactivity and turnover. Twf2a-deficient mice revealed an age-dependent macrothrombocytopenia that could be explained by a markedly decreased platelet half-life, likely due to the pronounced hyper-reactivity of \(Twf2a^{-/-}\) platelets. The latter was characterized by sustained integrin acti- vation and thrombin generation in vitro that translated into accelerated thrombus formation in vivo. To further elucidate mechanisms of integrin activation, Rap1-GTP-interacting adaptor molecule (RIAM)-null mice were generated. Despite the proposed critical role of RIAM for platelet integrin activation, no alterations in this process could be found and it was concluded that RIAM is dispensable for the activation of β1 and β3 integrins, at least in platelets. These findings change the current mechanistic understanding of platelet integrin activation.
Outside-in signaling by integrins and other surface receptors was supposed to regulate MK migration, but also the temporal and spatial formation of proplatelet protrusions. In this the- sis, phospholipase D (PLD) was revealed as critical regulator of actin dynamics and podo- some formation in MKs. Hence, the unaltered platelet counts and production in \(Pld1/2^{-/-}\) mice and the absence of a premature platelet release in the bone marrow of \(Itga2^{-/-}\) mice question the role of podosomes in platelet production and raise the need to reconsider the proposed inhibitory signaling by α2β1 integrins on proplatelet formation.
Non-muscle myosin IIA (NMMIIA) has been implicated as a downstream effector of the in- hibitory signals transmitted via α2β1 integrins. Besides Rho-GTPase signaling, also \(Mg^{2+}\) and transient receptor potential melastatin-like 7 (TRPM7) channel α-kinase are known regulators of NMMIIA activity. In this thesis, TRPM7 was identified as major regulator of \(Mg^{2+}\) homeostasis in MKs and platelets. Furthermore, decreased \([Mg^{2+}]_i\) led to deregulated NMMIIA activity and altered cytoskeletal dynamics that impaired thrombopoiesis and resulted in macrothrombocytopenia in humans and mice.
Das invasive Potential maligner Gliome beeinflusst maßgeblich die schlechte Prognose dieser Tumorentität. Migration und Invasion von Tumorzellen werden entscheidend durch die Cofilin-vermittelte Umstrukturierung des Aktin-Zytoskeletts geprägt, die durch die Aktivität antagonistischer Cofilin-Kinasen und -Phosphatasen reguliert wird.
Im Rahmen der vorliegenden Arbeit konnte ein progressiver Expressionsverlust der Cofilin-Phosphatase Chronophin mit ansteigendem Malignitätsgrad astrozytärer Gliome aufgezeigt werden, der mit einer Zunahme der Phosphorylierung von Cofilin einhergeht. In den entsprechenden Gewebeproben gelang gleichzeitig der Nachweis einer gesteigerten Expression der Cofilin-Kinase LIMK-2.
Genetische und epigenetische Analysen des Chronophin-Locus konnten eine Hypermethylierung im Bereich der Promotorregion der Phosphatase identifizieren, die möglicherweise dem Verlust von Chronophin in Glioblastom-Gewebeproben zugrunde liegt.
In Glioblastom-Zelllinien, die unterschiedliche Expressionsmuster von Chronophin aufwiesen, konnten hingegen keine molekularen Alterationen festgestellt werden.
Untersuchungen des Einflusses von ROCK- und LIMK-Inhibitoren auf Glioblastomzellen konnten ausgeprägte Veränderungen der Zellmorphologie dokumentieren, wobei erstmals die Induktion eines stellate cell-Phänotyps unter Einfluss des LIMK-Inhibitors BMS-5 beschrieben wird. Während ROCK- und LIMK-Inhibitoren keinen Einfluss auf die 2D-Motilität der Tumorzellen hatten, wiesen die Glioblastomzellen in Abhängigkeit ihrer basalen Cofilin-Aktivität eine verstärkte bzw. verminderte 3D-Invasivität auf.
Die Erkenntnisse dieser Arbeit unterstreichen die Bedeutung des Cofilin-Signalweges für die Migration und Invasion von Gliomzellen, zeigen neue Angriffspunkte in der Therapie maligner Gliome auf und warnen zugleich vor einem unkritischen Einsatz neuer Wirkstoffe.
Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer.
Still, most of the mammalian HAD phosphatases remain functionally uncharacterized.
This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos.
To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation.
The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells.
These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling.
Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase.
To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions.
Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under
hypoxic (~1% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3’-phosphate (GADP).
Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism.
Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.
Die Phosphatase PDXP (auch bekannt als Chronophin) gehört zur Familie der HAD Phosphatasen, einer ubiquitär exprimierten Enzymklasse mit wichtigen physiologischen Funktionen. PDXP zeigt Phosphatase-Aktivität gegenüber seinem Substrat Pyridoxal 5´-Phosphat (PLP), der aktivierten Form von Vitamin B6. PDXP-defiziente Mäuse (Knockout-Mäuse) weisen im Vergleich zu Wildtypen verdoppelte PLP-Konzentrationen in Erythrozyten sowie im Gesamthirn auf. Vermutlich kommt PDXP daher eine wichtige Funktion in Erythrozyten und im Hirn zu. Ziel dieser Arbeit war es, erste Einblicke in diese Funktion(en) von PDXP zu erlangen.
Hierzu wurden HPLC-basierte Analysen der erythrozytären PLP-Konzentrationen in Wildtyp- sowie PDXP-defizienten Mäusen durchgeführt. Dabei ließen sich die rund doppelt so hohen erythrozytären PLP-Level in den KO-Mäusen bestätigen. Zudem ist es gelungen, eine Methode zur Messung der endogenen Phosphatase-Aktivität von PDXP in Erythrozytenlysaten zu etablieren. So konnte im Wildtyp anhand der Verringerung der PLP-Konzentrationen pro Zeiteinheit eine erythrozytäre PDXP-Aktivität nachgewiesen werden. Dazu waren die Inkubation mit Pyridoxin, sowie die Anwendung eines Inhibitors der PDXK notwendig. Eine bis dato vermutete Funktion der PDXP, zur Mobilisation von erythrozytärem PLP während Fastenzeiten, konnte ausgeschlossen werden. So zeigte der Vergleich der erythrozytären PLP-Konzentrationen aus gefasteten mit normal gefütterten Tieren in beiden Genotypen exakt dieselbe prozentuale PLP-Verringerung. Während Nahrungszufuhr ließ sich jedoch eine Funktion der Phosphatase PDXP als „Converter“ von Pyridoxin zu Pyridoxal erkennen. Ausgehend von PN konnte im Wildtyp (über die Zwischenprodukte PNP und PLP) eine PDXP-abhängige Dephosphorylierung von PLP zu PL erfolgen. So wies der Wildtyp eine rund vierfach höhere PL-Produktion auf, verglichen mit der PDXP-defizienten Maus. Die Phosphatase PDXP erwies sich als essenziell für die erythrozytäre Konversion von Pyridoxin zu Pyridoxal. Dadurch erreicht der Organismus eine metabolische Flexibilität, die ihn bis zu einem gewissen Grad unabhängig von der Nahrungsauswahl macht. Zudem können Zellen oder Organe, denen durch das Fehlen der PNPO, die Konversion zu PLP nicht möglich ist, mit PL versorgt werden.
Aus der hohen Reaktivität von PLP mit umliegenden Nucleophilen ergibt sich eine gewisse Problematik für die Zelle im Umgang mit freiem PLP. So liegt der Großteil des erythrozytären PLPs gebunden an Proteine (vor allem Hämoglobin) vor. Anhand von Filtern (MWCO, 3000) ließ sich zwischen der hier definiert als „freien“ und der „gebundenen“ Form von PLP differenzieren. So konnten erste Erkenntnisse zur Rolle von PDXP als Determinator freier PLP-Konzentrationen in Erythrozyten und insbesondere im Hippocampus erlangt werden. Im Hippocampus ergaben sich insgesamt deutlich höhere Konzentrationen an freiem PLP als in den Erythrozyten und es bestand zudem ein Unterschied zwischen den Genotypen. So wiesen die KO-Mäuse ~1/3 höhere freie PLP-Konzentrationen im Vergleich zu den Wildtypen auf. Schließlich konnte ein Effekt des Tieralters auf den PLP-Metabolismus festgestellt werden. Sowohl in den Erythrozyten als auch im Hippocampus ergaben sich alterskorrelierte Änderungen ihrer PLP-Konzentrationen. Zudem zeigten Western Blot Analysen altersbedingte Unterschiede ihrer Vitamin B6-Enzymexpressionen. So wiesen ältere Wildtypen im Hippocampus eine fünffach erhöhte PDXP-Expression verglichen mit jüngeren Tieren auf. In den Erythrozytenlysaten hingegen zeigten ältere Tiere beider Genotypen eine rund vierfach geringere PNPO-Expression gegenüber jüngeren Tieren. Die mit dem Alter eintretende physiologische Verringerung der erythrozytären PNPO-Expression würde somit für den Organismus einen Verlust seiner metabolischen Flexibilität bedeuten, die mit der Konversion von PN zu PL einhergeht.
Platelets are small anucleated cell fragments that originate from megakaryocytes (MKs), which are large cells located in the bone marrow (BM). MKs extend long cytoplasmic protrusions, a process which is called proplatelet formation, into the lumen of the sinusoidal vessels where platelets are sized by the bloodstream. During the process of platelet biogenesis, segments of the MK penetrate the endothelium and, through cytoskeletal remodeling inside the MK, proplatelet fragments are released. Rho GTPases, such as RhoA and RhoB, are critically involved in cytoskeletal rearrangements of both the actin and the tubulin cytoskeleton.
The first part of this thesis concentrated on the protein RhoB and its involvement in cytoskeletal organization in MKs and platelets. Single knockout (KO) mice lacking RhoB had a minor microthrombocytopenia, which means a smaller platelet size and reduced platelet number, accompanied by defects in the microtubule cytoskeleton in both MKs and platelets. In particular, tubulin organization and stability, which is regulated by posttranslational modifications of α-tubulin, were disturbed in RhoB-/- platelets. In contrast, RhoB-/- MKs produced abnormally shaped proplatelets but had unaltered posttranslational modifications of α-tubulin.
The second part focused on the influence of RhoA and RhoB on MK localization and platelet biogenesis in murine BM. Many intact RhoA-/- MKs are able to transmigrate through the endothelial layer and stay attached to the vessel wall, whereas only 1% of wildtype (wt) MKs are detectable in the intrasinusoidal space. Concomitant deficiency of RhoA and RhoB reverts this transmigration and results in macrothrombocytopenia, MK clusters around the vessel in the BM and defective MK development. The underlying mechanism that governs MKs to distinct localizations in the BM is poorly understood, thus this thesis suggests that this process may be dependent on RhoB protein levels, as RhoA deficiency is coincided with increased RhoB levels in MKs and platelets.
The third part of this thesis targeted the protein PDK1, a downstream effector of Rho GTPases, in regard to MK maturation and polarization throughout thrombopoiesis. MK- and platelet-specific KO in mice led to a significant macrothrombocytopenia, impaired actin cytoskeletal reorganization during MK spreading and proplatelet formation, with defective MK maturation. This was associated with decreased PAK activity and, subsequently, phosphorylation of its substrates LIMK and Cofilin. Together, the observations of this thesis highlight the importance of Rho GTPases and their downstream effectors on the regulation of the MK and platelet cytoskeleton.
Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of enzymes with important functions in physiology and disease. HAD phosphatases can target diverse metabolites, lipids, DNA, and serine/threonine or tyrosine phosphorylated proteins with often high specificity (Seifried et al., 2013). These enzymes thus markedly enlarge the repertoire and substrate spectrum of mammalian phosphatases. However, the basis of HAD phosphatase substrate specificity is still elusive and a number of mammalian HAD phosphatases remain uncharacterized to date. This study characterizes the biochemical and structural properties of AUM (aspartate-based, ubiquitous, Mg2+-dependent phosphatase), a previously unexplored mammalian HAD phosphatase.
In vitro phosphatase assays of purified, recombinant AUM showed phosphatase activity towards para-nitrophenyl phosphate and adenine and guanine nucleotide di- and triphosphates. Inhibitor studies indicated that similar to other HAD superfamily members, the AUM-catalyzed dephosphorylation reaction proceeds via a pentacovalent phosphoaspartate intermediate. In line with an aspartate-based catalytic mechanism, AUM was insensitive to inhibitors of serine/threonine phosphatases. The characterization of the purified recombinant murine enzyme also revealed that AUM exists in equilibrium between dimers and tetramers.
AUM was identified as the closest, yet functionally distinct relative of chronophin, a pyridoxal 5’-phosphate and serine/threonine-directed phosphatase. Phylogenetic analyses showed that AUM and chronophin evolved via duplication of an ancestral gene at the origin of the vertebrates. In contrast to chronophin, AUM acts as a tyrosine-specific HAD-type phosphatase in vitro and in cells. To elucidate how AUM and chronophin achieve these distinct substrate preferences, comparative evolutionary analyses, biochemical approaches and structural analyses were combined. Swapping experiments of less homologous regions between AUM and chronophin were performed. The mutational analysis revealed residues important for AUM catalysis and specificity. A single differently conserved residue in the cap domain of AUM or chronophin is crucial for phosphatase specificity (AUML204, chronophinH182). The X-ray crystal structure of the AUM cap fused to the catalytic core of chronophin (CAC, PDB: 4BKM) was solved to 2.65 Å resolution. It presents the first crystal structure of the murine AUM capping domain. The detailed view of the catalytic clefts of AUM and chronophin reveals the structural basis of the divergent substrate specificities. These presented findings provide insights into the design principles of capped HAD phosphatases and show that their substrate specificity can be encoded by a small number of predictable residues. In addition, the catalytic properties of AUM were investigated, identifying a mechanism of reversible oxidation regulating the activity of AUM in vitro. AUM phosphatase activity is inhibited by oxidation and can be recovered by reduction. The underlying molecular mechanism was revealed by mutational analyses. The cysteines C35, C104 and C243, located in the AUM core domain, are responsible for the inhibition of AUM by oxidation. C293 mediates the redox-dependent tetramerization of AUM in vitro. Based on the chronophin and CAC structure, a direct impact of the oxidation of C35 on the nucleophile D34 is proposed. In addition, a redox-dependent disulfide bridge (C104, C243), connecting the core and cap domain of AUM may be important for an open/close-mechanism. This hypothesis is supported by CD spectroscopy experiments that demonstrate a structural change in AUM upon reduction. These data present the first evidence for the regulation of AUM catalysis by reversible oxidation. This finding is so far unique in the field of HAD phosphatases.
In this context, the first cell-based AUM activity assay was developed. For this, the artificial substrate pNPP was combined with the reducing agent DTT to create a specific AUM activity readout. This fractionation-based assay is the first tool to differentiate between cell lines or tissues with different AUM concentrations or activities.
Taken together, the presented biochemical characterization reveals the specificity determinants and catalytic properties of AUM. General insights into structural determinants of mammalian HAD phosphatase substrate recognition are provided and reversible oxidation as possible regulatory mechanism for AUM is proposed. These findings constitute a framework for further functional analyses to elucidate the biomedical importance of AUM.
In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work.
Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases.
Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity.
Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1.
Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.
The haloacid dehalogenase (HAD) family of phosphatases is an ancient, ubiquitous group of enzymes, and their emerging role in human health and disease make them attractive targets for detailed analyses.
This thesis comprises the biochemical and structural characterization of chronophin, an HAD-type
phosphatase, which has been shown to act on Ser3-phosphorylated cofiln-1, a key regulator of actin dynamics, and on the Ser/Thr-phosphorylated steroid receptor co-activator 3 (SRC-3). Besides being a specific phosphoprotein phosphatase, chronophin also acts on the small molecule pyridoxal 5'-phosphate (PLP, vitamin B6), implying that chronophin serves as a regulator of a variety important physiological pathways. The analysis of chronophin was performed on different levels, ranging from intrinsic regulatory mechanisms, such as the allosteric regulation via dimerization or the characterization of specificity determinants, to modes of extrinsic modulation, including the association with putative interacting proteins or the generation of chronophin-specific inhibitors.
The association of the previously identified putative chronophin interactors calcium- and integrinbinding protein 1 (CIB1) and calmodulin was investigated using recombinantly expressed and purified proteins. These studies revealed that the interaction of chronophin with CIB1 or calmodulin is mutually exclusive and regulated by calcium. Neither CIB1 nor calmodulin had an effect on the in vitro chronophin phosphatase activity towards PLP or phospho-cofilin-1, but might regulate other functions of this important phosphatase.
The role of chronophin dimerization was studied by generating a constitutively monomeric variant,
which showed reduced PLP hydrolyzing activity. X-ray crystallographic studies revealed that dimerization is essential for the positioning of the substrate specificity loop in chronophin, unraveling a previously unknown mechanism of allosteric regulation through a homophilic interaction. This mechanism potentially applies to other enzymes of the C2a subfamily of HAD-type phosphatases, as all structurally characterized members show a conserved mode of dimerization.
The general determinants of substrate specificity in the C2a subfamily of HAD phosphatases were
investigated by performing domain swapping experiments with chronophin and its paralog AUM and
subsequent biochemical analyses of the hybrid proteins. The X-ray crystallographic structure
determination of the chronophin catalytic domain equipped with the AUM capping domain revealed the first partial structure of AUM. This structural information was then used in subsequent studies that analyzed the divergent substrate specificities of AUM and chronophin in an evolutionary context.
Finally, a set of four chronophin inhibitors were generated based on the structure of PLP and
characterized biochemically, showing moderate inhibitory effects with IC50-values in the micromolar range. These compounds nevertheless constitute valuable tools for future in vitro experiments, such as studies concerning the structure-function relationship of chronophin as a PLP phosphatase. In addition, the crystal structure of one inhibitor bound to chronophin could be solved. These results provide the basis for the further development of competitive chronophin inhibitors with increased specificity and potency.
Im Rahmen dieser Doktorarbeit sollte anknüpfend an die Ergebnisse aus vo-rangegangenen Untersuchungen der AG Tessmer, das von Büchner et al. [1] vorgestellte Modell zur DNA-Schadenserkennung, welches im Speziellen auf Daten zu den Glykosylasen hTDG und hOGG1 basierte, auf seine Allgemein-gültigkeit für DNA-Glykosylasen untersucht werden. Das Modell beschreibt den Prozess der Schadenserkennung als eine notwendige Übereinstimmung der passiven Biegung am Schadensort mit dem aktiven BiegungswinkeI der scha-densspezifischen Glykosylase. Ein wesentlicher Bestandteil dieser Arbeit war zudem die Etablierung einer automatisierten Messsoftware zur objektiven Biegewinkelmessung an DNA-Strängen in rasterkraftmikroskopischen Aufnah-men. Dies wurde mit verschiedenen Bildverarbeitungsprogrammen sowie einer in MATLAB implementierten Messsoftware erreicht und das Programm zudem auf die Biegewinkelmessung von proteininduzierten Biegewinkeln erweitert. Zur Anwendung kam die Methode der automatisierten Biegewinkelmessung sowohl an rasterkraftmikroskopischen Aufnahmen der Glykosylase MutY gebunden an ungeschädigter DNA als auch an Aufnahmen von DNA mit und ohne Basen-schaden. Neben oxoG:A und G:A, den spezifischen MutY-Zielschäden, wurden auch andere Basenschäden wie beispielsweise oxoG:C und ethenoA:T vermes-sen und zudem die von der Glykosylase MutY an ungeschädigter DNA induzier-te Biegung mit den Biegewinkeln der jeweiligen Zielschäden verglichen. Die Übereinstimmung in den Konformationen der Zielschäden und der Reparatur-komplexe auch für die Glykosylase MutY (wie bereits für hTDG und hOGG1 in oben genannter Arbeit gezeigt) erlauben ein verbessertes Verständnis der Schadenssuche und -erkennung durch DNA-Glykosylasen, indem sie die All-gemeingültigkeit einer Biegungsenergie-basierten initialen Schadenserkennung durch DNA-Glykosylasen unterstützen. Die etablierte Messsoftware kann zu-künftig an weiteren DNA-Schäden und den entsprechenden Protein-DNA-Komplexen ihre Anwendung finden und kann somit durch die effektive Gewin-nung objektiver Daten in großer Menge zur Stützung des Modells beitragen.
Abstract
Glioblastomas, primary brain tumors, represent a tumor entity with a dismal prognosis and a median survival of only about one year. Invasion into the healthy brain parenchyma contributes substantially to the malignancy of this type of brain tumor. Therefore, a better understanding of the mechanisms promoting the invasive behavior of these brain tumors is needed to identify new therapeutic targets.
Cofilin, an actin regulatory protein, has been shown to be an important regulator of the invasive behavior of tumor cells in other types of cancer and the actin cytoskeleton is involved in the formation of a variety of cellular structures important for cell migration and invasion. Cofilin is regulated by phosphorylation on a single residue, serine 3. The aim of this thesis was to examine the role of the cofilin regulatory phosphatase chronophin for glioma cell migration and invasion.
First, it was established that chronophin depletion in the cell line GBM6840 leads to an increase in the ratio of phosphorylated cofilin to total cofilin. Higher chronophin levels were correlated with a decrease in F-actin in the cell lines GBM6840 and U87 as measured in an actin spin down assay and in a flow cytometry based assay.
Furthermore, it was shown that knockdown of chronophin in two different cell lines, GBM6840 and DBTRG-05-MG, strongly increased their invasiveness in vitro. Expression of human chronophin in the cell line U87 decreased its invasiveness substantially. There was no difference in cell proliferation between GBM6840 and DBTRG-05-MG cells expressing a chronophin targeting shRNA or a control shRNA and U87 cells transfected with an empty vector or a human chronophin encoding plasmid. The increase in invasiveness after chronophin depletion could be correlated with an increase in directionality in cell migration under 2D culture conditions in the cell lines U87 and GBM6840. Moreover, treatment with the ROCK inhibitor Y-27632 decreased directionality in GBM6840 cells under 2D culture conditions and reduced the invasiveness of GBM6840 chronophin shRNA cells back to control levels.
Expression of a non-phosphorylatable cofilin mutant, the S3A mutant, was able to reduce invasiveness and to reduce directionality under 2D culture conditions back to control levels in GBM6840 chronophin shRNA cells.
This provides important evidence for the involvement of cofilin phosphoregulation in the phenotypes described above.
In vivo, when injected into NOD-SCID mice, chronophin depleted cells showed a dramatic growth reduction as compared to control and rescue cells.
Transciptomic characterization of GBM6840 cells by microarray analysis and subsequent comparison of the data with microarray profiles of normal brain tissues and different glioma entities identified two specifically chronophin regulated transcripts potentially involved in tumor progression and invasion, MXI1 and EDIL3. Moreover, c-myc was identified as a significantly altered transcription factor after chronophin deregulation based on the number of c-myc target molecules in the microarray dataset.
MXI1 is a potential negative regulator of c-myc dependent transcription, and was strongly downregulated after chronophin knockdown in GBM6840. In line with this, the activity of a c-myc reporter plasmid was increased after chronophin depletion in GBM6840 and reduced after chronophin expression in U87 cells.
However, the protein level of the c-myc protein was reduced after chronophin depletion in GBM6840.
Finally, anaylsis of the expression of proteases known to be important for glioblastoma pathogenesis revealed no major changes in protease expression between chronophin depleted and control cells.
Therefore, a comprehensive analysis of chronophin in the context of glioma pathogenesis has been performed in this thesis. It has been shown that chronophin depletion strongly enhanced invasiveness of glioma cells and that it induced transcriptomic changes potentially involved in tumor progression. The proteins regulating cofilin phosphorylation are therefore valuable therapeutic targets for anti-invasive therapy in glioblastomas. Inhibitors for kinases upstream of cofilin, e.g. LIMKs and ROCKs, are available, and might be promising agents for anti-invasive therapy.
In dieser Arbeit geht es um die Phosphoglykolatphosphatase (PGP), die als Phosphatase vom Haloazid Dehalogenase-Typ (HAD-Phosphatase) zu der ubiquitär vorkommenden Superfamilie der HAD-Hydrolasen gehört. In der Literatur ist eine in vitro Phosphatase-Aktivität gegenüber 2-Phospho-L-Laktat (2PL), 4-Phospho-D-Erythronat (4PE), Phosphoglykolat (PG) und Glycerol-3-Phosphat (G3P) beschrieben. 2PL und 4PE entstehen in Nebenreaktionen während der Glykolyse und hemmen bei Akkumulation die Glykolyse bzw. den Pentosephosphatweg. PG kann auch in einer Nebenreaktion während der Glykolyse oder im Rahmen der Reparatur von oxidativen DNA-Schäden entstehen. G3P entsteht aus Dihydroxyacetonphosphat und bildet das Kohlenhydratgerüst der Triacylglyceride (TAG). Zelluläre Studien konnten Hinweise auf die Regulierung des epidermalen wachstumsfaktor-(EGF-)induzierten Zytoskelettumbaus durch die PGP liefern und die Untersuchung von Mäusen mit PGP-Inaktivierung zeigte einen Einfluss auf die Zellproliferation und embryonale Entwicklung. Die Regulation der PGP-Expression führte zu Veränderungen im Kohlenhydrat- und Fettstoffwechsel.
Die Untersuchung der PGP-Funktionen erfolgte bislang ausschließlich mit genetischen Ansätzen. Aufgrund von möglichen Kompensationsmechanismen und Off-Target-Effekten müssen genetische und pharmakologische Methoden als sich ergänzende Ansätze verstanden werden. Um die Funktionen der PGP besser zu verstehen, fokussiert sich die vorliegende Arbeit auf die gezielte pharmakologische PGP-Inhibition. In Vorarbeiten wurden 41.000 Moleküle gescreent und fünf potentielle Inhibitoren identifiziert. Ziele dieser Arbeit waren zum einen die Implementierung der Inhibitor # 1-Behandlung in der Zellkultur, zum anderen die Charakterisierung der PGP-Hemmung durch Inhibitor # 48 und die Durchführung erster Selektivitätstestungen mit Inhibitor # 48.
Zusammenfassend kann festgehalten werden, dass Inhibitor # 1 in der Lage ist, die endogene PGP in Zelllysaten der murinen spermatogonialen Zelllinie (GC1) zu hemmen. Unter bestimmten Bedingungen führte die Inhibitor # 1-Behandlung der GC1-Zellen zur Hemmung der PGP. Erste Analysen zellulärer Inhibitoreffekte konnten eine Steigerung der TAG-Konzentration in behandelten GC1-Zellen nachweisen. Die PGP-Hemmung durch Inhibitor # 48 wurde als unkompetitive Inhibition charakterisiert und es zeigten sich keine relevanten Inhibitoreffekte auf die HAD-Phosphatasen Magnesium-abhängige Phosphatase 1 (MDP1), Lysin-Histidin-Pyrophosphat-Phosphatase (LHPP) und Polynukleotidase 5'-Kinase/3'-Phosphatase (PnkP). Dagegen konnte eine Aktivitätssteigerung von Phospho 2 beobachtet werden. Die vorliegende Arbeit liefert somit erste Erkenntnisse über die Anwendung des PGP-Inhibitors # 1 in der Zellkultur und schafft die Grundlage für nachfolgende Untersuchungen mit Inhibitor # 48. Weitere Experimente sind notwendig, die die Inhibitorbehandlung in der Zellkultur optimieren und die Selektivität weiter charakterisieren, um mithilfe der Inhibitoren neue Erkenntnisse über die physiologische und pathophysiologische Rolle der PGP gewinnen zu können.
Die Pyridoxal-5‘-Phosphat Phosphatase (PDXP), auch bekannt als Chronophin (CIN), ist eine HAD-Phosphatase, die beim Menschen ubiquitär exprimiert wird und eine entscheidende Rolle im zellulären Vitamin-B6-Metabolismus einnimmt. PDXP ist in der Lage Pyridoxal-5‘-Phosphat (PLP), die co-enzymatisch aktive Form von Vitamin B6, zu dephosphorylieren. In-vivo Studien mit Mäusen zeigten, dass die Abwesenheit von PDXP mit verbesserten kognitiven Leistungen und einem verringerten Wachstum von Hirntumoren assoziiert ist. Dies begründet die gezielte Suche nach einem pharmakologischen Inhibitor für PDXP. Ein Hochdurchsatz-Screen legte nahe, dass 7,8-Dihydroxyflavon (7,8-DHF) hierfür ein potenzieller Kandidat ist. Zahlreiche Studien beschreiben bereits vielfältige positive neurologische Effekte nach in-vivo Administration von 7,8-DHF, allerdings bleibt der genaue Wirkmechanismus umstritten und wird bis dato nicht mit PDXP in Zusammenhang gebracht. Ziel dieser Arbeit ist es, die Inhibition von PDXP durch 7,8-DHF näher zu charakterisieren und damit einen Beitrag zur Beantwortung der Frage zu leisten, ob PDXP an den 7,8-DHF-induzierten Effekten beteiligt ist.
Hierzu wurde der Effekt von 7,8-DHF auf die enzymatische Aktivität von rekombinant hergestelltem, gereinigtem PDXP in in-vitro Phosphatase-Assays charakterisiert. Um die Selektivität von 7,8-DHF gegenüber PDXP zu untersuchen, wurden fünf weitere HAD-Phosphatasen getestet. Unter den analysierten Phosphatasen zeigte einzig die dem PDXP nah verwandte Phosphoglykolat Phosphatase (PGP) eine geringer ausgeprägte Sensitivität gegen 7,8-DHF. Ein Vergleich von 7,8-DHF mit sechs strukturell verwandten, hydroxylierten Flavonen zeigte, dass 7,8-DHF unter den getesteten Substanzen die höchste Potenz und Effektivität aufwies. Außerdem wurde eine Co-Kristallisation von PDXP mit 7,8-DHF durchgeführt, deren Struktur bis zu einer Auflösung von 2,0 Å verfeinert werden konnte. Die in der Kristallstruktur identifizierte Bindungsstelle von 7,8-DHF an PDXP wurde mittels verschiedener, neu generierter PDXP-Mutanten enzymkinetisch bestätigt. Zusammenfassend zeigen die hier beschriebenen Ergebnisse, dass 7,8-DHF ein direkter, selektiver und vorwiegend kompetitiver Inhibitor der PDXP-Aktivität ist, mit einer IC50 im submikromolaren Bereich.
Die Ergebnisse dieser in-vitro Untersuchungen motivieren zu weiterer Forschung bezüglich der 7,8-DHF-vermittelten Inhibition der PDXP-Aktivität in Zellen, um die Frage beantworten zu können, ob PDXP auch in-vivo ein relevantes Target für 7,8-DHF darstellt.
Mammalian phoshoglycolate phosphatase (PGP, also known as AUM) belongs to the ubiquitous HAD superfamily of phosphatases. As several other members of HAD phosphatases, the Mg2+-dependent dephosphorylation is conducted via a nucleophilic attack from a conserved aspartate residue in the catalytic cleft. The protein structure of PGP could not yet be solved entirely. Only a hybrid consisting of the PGP cap and the PDXP core (pyridoxal phosphatase, closest enzyme paralog) was crystallizable so far. PGP is able to efficiently dephosphorylate 2-phosphoglycolate, 2-phospho-L-lactate, 4-phospho-D-erythronate, and glycerol-3-phosphate in vitro which makes them likely physiological substrates. The first three substrates can be derived from metabolic side reactions (during glycolysis) and inhibit key enzymes in glycolysis and pentose phosphate pathway, the latter is situated at the intersection between glycolysis and lipogenesis. 2-phosphoglycolate can also be released in the context of repair of oxidative DNA damage. The activity of purified PGP can be reversibly inhibited by oxidation - physiologically likely in association with epidermal growth factor (EGF) signal transduction. In fact, an association between persistently lacking PGP activity (via downregulation) and the presence of hyperphosphorylated proteins after EGF stimulation has been identified. Reversible oxidation and transient inactivation of PGP may be particularly important for short-term and feedback regulatory mechanisms (as part of the EGF signaling). Furthermore, cellular proliferation in PGP downregulated cells is constantly reduced. Whole-body PGP inactivation in mice is embryonically lethal. Despite the many well-known features and functions, the knowledge about PGP is still incomplete.
In the present work the influence of reactive oxygen species (ROS) on PGP activity in cells und a possible connection between oxidative stress and the proliferation deficit of PGP downregulated cells was investigated. For the experiments, a spermatogonial cell line was used (due to the high PGP expression in testis). PGP activity can be reversibly inhibited in cellular lysates by H2O2 (as a ROS representative). Reversible oxidation could thus indeed be physiologically important. More oxidative DNA damage (by bleomycin) showed no PGP-dependent effects here. EGF stimulation (as an inducer of transient and well-controlled ROS production), low concentrations of menadione (as an oxidant) and N-acetylcysteine (as an antioxidant) were able to approximate the proliferation rate in PGP downregulated cells to that of control cells. The redox regulation of PGP could thus have an influence on cellular proliferation as a feedback mechanism - a mechanism that could not take place in PGP downregulated cells. However, the connections are probably even more complex and cannot be elucidated by a sole examination of the proliferation rate. The present results can thus only be regarded as preliminary experiments.
For a better understanding of the features and functions of PGP, this work then focused on specific regulation of enzyme activity by pharmacologically applicable small molecules. Four potent inhibitors had previously been identified in a screening campaign. In this work, three of these four inhibiting compounds could be further characterized in experiments with highly purified, recombinant murine and human PGP. Compounds #2 and #9 showed competitive inhibition properties with a markedly rising KM value with little or no change in vmax. The results were consistent for all tested protein variants: the murine and the human PGP as well as a PGP/PDXP hybrid protein. Compound #1 was the most potent and interesting PGP-inhibitory molecule: less change in KM and a constant decrease in vmax as well as a lower impact on the PGP/PDXP hybrid hint at a mixed mode of inhibition as a combination of competitive and non-competitive inhibition. The characterization of the potential inhibitors can serve as a basis for further structural analysis and studies on the complex physiological role of PGP.
Adipocytes are specialized cells found in vertebrates to ensure survival in terms of adaption to food deficit and abundance. However, their dysfunction accounts for the pathophysiology of metabolic diseases such as T2DM. Preliminary data generated by Mona Löffler suggested that PKD1 is involved in adipocyte function. Here, I show that PKD1 expression and activity is linked to lipid metabolism of murine adipocytes. PKD1 gene expression and activity was reduced in murine white adipose tissue upon fasting, a physiological condition which induces lipolysis. Isoproterenol-stimulated lipolysis in adipose tissue and 3T3-L1 adipocytes reduced PKD1 gene expression. Silencing ATGL in adipocytes inhibited isoproterenol-stimulated lipolysis, however, the β-adrenergic
stimulation of ATGL-silenced adipocytes lowered PKD1 expression levels as well. Adipose tissue of obese mice exhibited high PKD1 RNA levels but paradoxically lower protein levels of phosphorylated PKD1-Ser916. However, HFD generated a second
PKD1 protein product of low molecular weight in mouse adipose tissue. Furthermore, constitutively active PKD1 predominantly displayed nuclear localization in 3T3-L1 adipocytes containing many fat vacuoles. However, adipocytes
overexpressing non-functional PKD1 contained fewer lipid droplets and PKD1-KD was distributed in cytoplasm. Most importantly, deficiency of PKD1 in mouse adipose tissue caused expression of genes involved in adaptive thermogenesis such as UCP-1 and thus generated brown-like phenotype adipocytes. Thus, PKD1 is implicated in adipose tissue function and presents an interesting target for therapeutic approaches in the prevention of obesity and associated diseases.
Measles is an extremely contagious vaccine-preventable disease responsible
for more than 90000 deaths worldwide annually. The number of deaths has
declined from 8 million in the pre-vaccination era to few thousands every year due
to the highly efficacious vaccine. However, this effective vaccine is still unreachable
in many developing countries due to lack of infrastructure, while in developed
countries too many people refuse vaccination. Specific antiviral compounds are not
yet available. In the current situation, only an extensive vaccination approach
along with effective antivirals could help to have a measles-free future. To develop
an effective antiviral, detailed knowledge of viral-host interaction is required.
This study was undertaken to understand the interaction between MV and
the innate host restriction factor APOBEC3G (A3G), which is well-known for its
activity against human immunodeficiency virus (HIV). Restriction of MV
replication was not attributed to the cytidine deaminase function of A3G, instead,
we identified a novel role of A3G in regulating cellular gene functions. Among two
of the A3G regulated host factors, we found that REDD1 reduced MV replication,
whereas, KDELR2 hampered MV haemagglutinin (H) surface transport thereby
affecting viral release. REDD1, a negative regulator of mTORC1 signalling
impaired MV replication by inhibiting mTORC1. A3G regulated REDD1
expression was demonstrated to inversely correlate with MV replication. siRNA
mediated silencing of A3G in primary human blood lymphocytes (PBL) reduced
REDD1 levels and simultaneously increased MV titres. Also, direct depletion of
REDD1 improved MV replication in PBL, indicating its role in A3G mediated
restriction of MV. Based on these finding, a new role of rapamycin, a
pharmacological inhibitor of mTORC1, was uncovered in successfully diminishing
MV replication in Vero as well as in human PBL. The ER and Golgi resident
receptor KDELR2 indirectly affected MV by competing with MV-H for cellular
chaperones. Due to the sequestering of chaperones by KDELR2, they can no longer
assist in MV-H folding and subsequent surface expression. Taken together, the two
A3G-regulated host factors REDD1 and KDELR2 are mainly responsible for
mediating its antiviral activity against MV.