110 research outputs found
Genexpressionsanalyse an humanen Neuroblastomzellen nach 6-Hydroxydopamin-Behandlung
Morbus Parkinson ist eine chronische neurodegenerative Erkrankung, die durch einen Verlust der dopaminergen (DA) Neurone in der Substantia nigra pars compacta, gekennzeichnet ist. Die Ătiologie ist bisher weitgehend unbekannt. Die Behandlung von Nervenzellen mit 6-Hydroxydopamin (6-OHDA) ist ein etabliertes in vivo und in vitro Modell, um die Auswirkungen des oxidativen Stresses in M. Parkinson Gehirnen nachzuahmen. Um die nachgeschalteten Ereignisse in Zellen nach Exposition mit 6-OHDA zu charakterisieren, haben wir die dopaminerge humane Neuroblastomzell-Linie (SH-SY5Y Zellen) mit 6-OHDA behandelt und anschlieĂend das Profil der Genexpression mittels der reversen arbitrĂ€ren geprimten Polymerasen-Kettenreaktion (RAP-PCR) ermittelt. Mit der RAP-PCR können die Unterschiede in der Genexpression zwischen zwei oder mehreren Proben ermittelt werden. Reproduzierbare Unterschiede in der Genexpression der mit 6-OHDA behandelten Zellen (die Behandlung erfolgte mit 50ÎŒM bzw. 100ÎŒM 6-OHDA ĂŒber 24h) zeigten sich im Vergleich zu unbehandelten Kontrollen. Von diesen wiesen 23 Sequenzen eine hochsignifikante Homologie zu bekannten humanen Kodierungssequenzen auf. Die Ergebnisse wurden mittels RT-PCR, Real-time PCR und Western-Blot Analyse bestĂ€tigt. In vier FĂ€llen [Tumoregulin-1 (TMEFF-1), Collapsin response mediator protein 1 (CRMP-1), Neurexin-1 und Phosphoribosylaminoimidazolsynthetase (GART)] wurde eine suppressive Regulation auf mRNA- sowie auf Proteinebene festgestellt. Dies bestĂ€tigte, dass die Regulierung, die sich in der RAP-PCR zeigte, nicht nur auf die Transkriptionsebene beschrĂ€nkt ist, sondern sich auch auf die entsprechende Proteinebene erstreckt. Es bestĂ€tigt sich somit, dass die von uns angewandte RAP-PCR als Methode zur Analyse der differentiellen Genexpression von SH-SY5Y im 6-OHDA-SchĂ€digungsmodell in vitro geeignet ist. Diese vier Proteine, die z.T. erstmalig in Bezug in dem ToxizitĂ€tsmodell identifiziert wurden, konnten als Proteine charakterisiert werden, die auch physiologisch eine Rolle im Zelluntergang Substantia nigra Neuronen spielen könnten. Weitere Studien fĂŒr die physiologische Rolle der identifizierten Gene und ihren Einfluss auf die Neurodegeneration im M. Parkinson sind allerdings nötig
Doxorubicin induces caspase-mediated proteolysis of KV7.1
Strigli A, Raab C, Hessler S, et al. Doxorubicin induces caspase-mediated proteolysis of KV7.1. Communications Biology. 2018;1(1): 155.Kv7.1 (KCNQ1) coassembles with KCNE1 to generate the cardiac IKs-channel. Gain- and loss-of-function mutations in KCNQ1 are associated with cardiac arrhthymias, highlighting the importance of modulating IKs activity for cardiac function. Here, we report proteolysis of Kv7.1 as an irreversible posttranslational modification. The identification of two C-terminal fragments of Kv7.1 led us to identify an aspartate critical for the generation of one of the fragments and caspases as responsible for mediating proteolysis. Activating caspases reduces Kv7.1/KCNE1 currents, which is abrogated in cells expressing caspase-resistant channels. Enhanced cleavage of Kv7.1 can be detected for the LQT mutation G460S, which is located adjacent to the cleavage site, whereas a calmodulin-binding-deficient mutation impairs cleavage. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provokes caspase-mediated cleavage of endogenous IKs in human cardiomyocytes. In summary, caspases are novel regulatory components of IKs channels that may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity
Impact of the first COVID lockdown on accident- and injury-related pediatric intensive care admissions in Germany - a multicenter study
Childrenâs and adolescentsâ lives drastically changed during COVID lockdowns worldwide. To compare accident- and injury-related admissions to pediatric intensive care units (PICU) during the first German COVID lockdown with previous years, we conducted a retrospective multicenter study among 37 PICUs (21.5% of German PICU capacities). A total of 1444 admissions after accidents or injuries during the first lockdown period and matched periods of 2017â2019 were reported and standardized morbidity ratios (SMR) were calculated. Total PICU admissions due to accidents/injuries declined from an average of 366 to 346 (SMR 0.95 (CI 0.85â1.05)). Admissions with trauma increased from 196 to 212 (1.07 (0.93â1.23). Traffic accidents and school/kindergarten accidents decreased (0.77 (0.57â1.02 and 0.26 (0.05â0.75)), whereas household and leisure accidents increased (1.33 (1.06â1.66) and 1.34 (1.06â1.67)). Less neurosurgeries and more visceral surgeries were performed (0.69 (0.38â1.16) and 2.09 (1.19â3.39)). Non-accidental non-suicidal injuries declined (0.73 (0.42â1.17)). Suicide attempts increased in adolescent boys (1.38 (0.51â3.02)), but decreased in adolescent girls (0.56 (0.32â0.79)). In summary, changed trauma mechanisms entailed different surgeries compared to previous years. We found no evidence for an increase in child abuse cases requiring intensive care. The increase in suicide attempts among boys demands investigation
Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) is involved in lysosomal cholesterol export
The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol - like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.Peer reviewe
Combined Fluorescence-Guided Resection and Intracavitary Thermotherapy with Superparamagnetic Iron-Oxide Nanoparticles for Recurrent High-Grade Glioma: Case Series with Emphasis on Complication Management
Background: Concepts improving local tumor control in high-grade glioma (HGG) are desperately needed. The aim of this study is to report an extended series of cases treated with a combination of 5-ALA-fluorescence-guided resection (FGR) and intracavitary thermotherapy with superparamagnetic iron oxide nanoparticles (SPION). Methods: We conducted a single-center retrospective review of all recurrent HGG treated with FGR and intracavitary thermotherapy (n = 18). Patients underwent six hyperthermia sessions in an alternating magnetic field and received additional adjuvant therapies on a case-by-case basis. Results: Nine patients were treated for first tumor recurrence; all other patients had suffered at least two recurrences. Nine patients received combined radiotherapy and thermotherapy. The median progression-free survival was 5.5 (95% CI: 4.67–6.13) months and median overall survival was 9.5 (95% CI: 7.12–11.79) months. No major side effects were observed during active treatment. Thirteen patients (72%) developed cerebral edema and more clinical symptoms during follow-up and were initially treated with dexamethasone. Six (33%) of these patients underwent surgical removal of nanoparticles due to refractory edema. Conclusions: The combination of FGR and intracavitary thermotherapy with SPION provides a new treatment option for improving local tumor control in recurrent HGG. The development of cerebral edema is a major issue requiring further refinements of the treatment protocol
Structural Determinants of M-Type KCNQ (Kv7) K+ Channel Assembly
Schwake M. Structural Determinants of M-Type KCNQ (Kv7) K+ Channel Assembly. Journal of Neuroscience. 2006;26(14):3757-3766.The ability of KCNQ (Kv7) channels to form hetero-oligomers is of high physiological importance, because heteromers of KCNQ3 with KCNQ2 or KCNQ5 underlie the neuronal M-current, which modulates neuronal excitability. In KCNQ channels, we recently identified a C-terminal subunit interaction (si) domain that determines their subunit-specific assembly. Within this si domain, there are two motifs that comprise âŒ30 amino acid residues each and that exhibit a high probability for coiled-coil formation. Transfer of the first or the second coiled-coil (TCC) domain from KCNQ3 into the KCNQ1 scaffold resulted in chimeras KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3, both of which coimmunoprecipitated with KCNQ2. However, only KCNQ1(TCC2)Q3 enhanced KCNQ2 currents and surface expression or exerted a strong dominant-negative effect on KCNQ2. Deletion of TCC2 within KCNQ2 yielded functional homomeric channels but prevented the current augmentation measured after coexpression of KCNQ2 and KCNQ3. In contrast, deleting TCC1 within KCNQ2 did not give functional homomeric KCNQ2 or heteromeric KCNQ2/KCNQ3 channels. Mutations that disrupted the predicted coiled-coil structure of TCC1 in KCNQ2 or KCNQ3 abolished channel activity after expressing these constructs singly or in combination, whereas helix-breaking mutations in TCC2 of KCNQ2 gave functional homomeric channels but prevented the heteromerization with KCNQ3. In contrast, KCNQ3 carrying a coiled-coil disrupting mutation in TCC2 hetero-oligomerized with KCNQ2.
Our data suggest that the TCC1 domains of KCNQ2 and KCNQ3 are required to form functional homomeric as well as heteromeric channels, whereas both TCC2 domains facilitate an efficient transport of heteromeric KCNQ2/KCNQ3 channels to the plasma membrane
Protein trafficking of members of the CLC and KCNQ ion channels
Titel
Inhaltsverzeichnis
1
1 Einleitung
3
2 Material
29
3 Methoden
34
4 Ergebnisse
57
5 Diskussion
93
6 AbkĂŒrzungen
109
7 Literaturverzeichnis
111
8 Anhang
123Mutationen in den Genen fĂŒr die KaliumkanĂ€le KCNQ2 und KCNQ3 fĂŒhren zu einem
vererbbaren epileptischen Syndrom mit der englischen Bezeichnung benign
familial neonatal convulsions (BFNC). Diese beiden spannungsabhÀngigen
KaliumkanĂ€le kommen fast ausschlieĂlich im ZNS vor und ihre Expressionsmuster
stimmen im Gehirn sehr stark ĂŒberein. KCNQ2 und KCNQ3 bilden Heteromere mit
neuen biophysikalischen Eigenschaften und vermitteln wahrscheinlich den
physiologisch sehr wichtigen M-Strom, der die neuronale Erregbarkeit
moduliert. Im Rahmen dieser Arbeit wurden die Ursachen fĂŒr den Anstieg des
Stroms untersucht, den man bei Koexpression von KCNQ2 und KCNQ3 in Xenopus
Oozyten im Verglich zu den jeweiligen homomeren KanÀlen beobachten kann. Dazu
wurden die EinzelkanalleitfÀhigkeiten und Offenwahrscheinlichkeiten (popen)
von homomeren KCNQ2 und KCNQ3 und heteromeren KCNQ2/KCNQ3 KanÀlen bestimmt.
Die EinzelkanalleitfÀhigkeiten und popen von homomeren KCNQ2 (i» 18pS, popen»
0,61) und heteromeren KCNQ2/KCNQ3 (i» 16pS, popen» 0,72) sind annÀhernd
gleich. Die EinzelkanalleitfÀhigkeit von KCNQ3 (i» 7,3) ist im Vergleich
ungefÀhr nur halb so groà und die Offenwahrscheinlichkeit (popen» 0,42) etwas
kleiner. Die Zunahme des Stroms bei Koexpression der beiden KCNQ-KanÀle konnte
somit nicht mit diesen Parametern erklÀrt werden. Koexpression von KCNQ3 mit
KCNQ2 erhöhte die OberflÀchenexpression von KCNQ2 um einen Faktor von ungefÀhr
fĂŒnf. Umgekehrt wurde die OberflĂ€chenexpression von KCNQ3 durch KCNQ2 um einen
Faktor von ungefÀhr zehn stimuliert. Eine KCNQ2 Mutante, die den C-Terminus
des Kanals verkĂŒrzt, erreichte nicht die ZelloberflĂ€che und konnte auch nicht
die OberflÀchenexpression von KCNQ3 erhöhen. Hingegen verhielten sich
Missense-Mutationen in der Porenregion und in der TransmembrandomÀne S6 von
KCNQ2 und KCNQ3 in ihrer OberflÀchenexpression annÀhernd wie die jeweiligen
Wildtyp KanÀle. Zusammenfassend kann also festgehalten werden, daà der Anstieg
des Stroms bei Koexpression von KCNQ2 und KCNQ3 vorwiegend durch eine erhöhte
Anzahl aktiver KanÀle in der Plasmamembran verursacht wird. Die Interaktion
und damit die Zielsteuerung der homo- und heteromeren KanÀle ist abhÀngig von
einem intakten C-Terminus. ClC-5 kommt hauptsÀchlich in den endozytotischen
Vesikeln des proximalen Tubulus der Niere vor, fĂŒr deren effiziente AnsĂ€uerung
er maĂgeblich verantwortlich ist. ClC-5 defiziente MĂ€use weisen eine stark
verminderte Endozytose (sowohl rezeptorvermittelt als auch aus der
FlĂŒssigphase) in der apikalen Region des proximalen Tubulus auf. Mutationen
innerhalb des CLCN5 Gens sind die Ursache der Dent`schen Erkrankung, deren
Hauptsymptome das Ausscheiden von Proteinen niederen Molekulargewichtes mit
dem Urin und die Bildung von Nierensteinen sind. In der vorliegenden Arbeit
wurde nach Endozytosemotiven innerhalb der AminosÀuresequenz von ClC-5
gesucht, um deren EinfluĂ auf die Zielsteuerung des Proteins zu bestimmen.
Dabei wurden zwei Internalisierungsmotive identifiziert, ein PY Motiv und ein
YF Motiv. Das PY Motiv konnte mit verschiedenen Methoden charakterisiert
werden, fĂŒr das YF Motiv steht eine derartige Analyse noch aus. Mutationen
innerhalb des PY Motivs erhöhten den ClC-5 vermittelten Strom und die
OberflÀchenexpression um einen Faktor von ungefÀhr zwei. Die Analyse der
Halbwertszeit des Wildtyps und des Kanals ohne funktionelles PY Motiv in der
Plasmamembran zeigte, daĂ die Halbwertszeit der Mutante gegenĂŒber dem Wildtyp
verlÀngert ist. Dieses Ergebnis deutete darauf hin, daà es sich bei dem PY
Motiv um ein Signal handelt, daĂ die Degradation des Kanalproteins vermittelt.
UnterstĂŒtzung findet diese SchluĂfolgerung durch Koexpressionsexperimente mit
WWP2 und nicht-funktionellen Konstrukten dieses Proteins. Dominant-negative
Mutanten des WWP2 Proteins erhöhen den ClC-5 vermittelten Strom zweifach,
allerdings nur, solange das PY Motiv intakt ist. Da es sich bei dem WWP2
Protein um eine Ubiquitin-Protein Ligase handelt, ist es wahrscheinlich, daĂ
entweder ClC-5 oder ein drittes, noch unbekanntes Protein ubiquitiniert wird.
ZusÀtzlich konnte im Rahmen dieser Arbeit gezeigt werden, daà ClC-5
vermittelte Ströme abnehmen, wenn die Endozytose durch rab5 oder rab5 Q79L in
Xenopus Oozyten stimuliert wird. Umgekehrt nahmen diese Ströme bei Inhibition
der Endozytose durch rab5 S34N zu. Die Zerstörung des PY Motivs hatte zur
Folge, daĂ die Ströme nicht mehr von der Endozytoserate beeinfluĂt werden
konnten. Zusammenfassend kann also festgehalten werden, daĂ sich ein
Endozytosemotiv im C-Terminus von ClC-5 befindet, das die Degradation des
Proteins vermittelt bzw. beeinfluĂt. Die vorliegenden Studien fĂŒhren also zu
einer Erweiterung unserer Kenntnisse ĂŒber Zielsteuerung und Protein-Protein-
Interaktionen von physiologisch sehr wichtigen Mitgliedern der CLC- und der
KCNQ-Ionenkanalfamilie.Mutations in either KCNQ2 or KCNQ3 cause benign familial neonatal convulsions
(BFNC), an inherited epilepsy of newborns. Both proteins are co-expressed in
the brain and associate to form heteromeric potassium channels. They are
thought to give rise to the so called M-currents, which are important
regulators of neuronal excitability. In this work the basis for the current
increase seen upon co-expression of both KCNQ subunits in Xenopus oocytes was
investigated. Therefore the single-channel conductances and open-probabilities
of homo- and heteromeric KCNQ2 and KCNQ3 channels were determined ((KCNQ2: i»
18pS, popen» 0,61) (KCNQ2/KCNQ3: i» 16pS, popen» 0,72) (KCNQ3: i » 7,3, popen»
0,42)). The mean single channel conductance and popen of heteromeric channels
were not significantly different from those of homomeric KCNQ2 channels,
whereas the conductance of KCNQ3 was about half that value. This indicates
that the increase of currents observed upon co-expressing both subunits in
Xenopus oocytes cannot be explained by an increase in these parameters. Co-
expression with KCNQ3 increased the surface expression of KCNQ2 by a factor of
5, and co-expression of KCNQ3 with KCNQ2 led to a >10fold increase of KCNQ3
surface expression. A KCNQ2 mutant associated with BFNC that truncates the
cytoplasmic carboxyterminus neither reached the surface nor stimulated KCNQ3
surface expression. By contrast, BFNC missense mutations in the transmembrane
regions of KCNQ2 or KCNQ3 left surface expression unchanged. Thus, the
increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly
due to an increase in surface expression, which is dependent on an intact
carboxyterminus. In the kidney ClC-5 chloride channel resides mainly in
vesicles of the endocytotic pathway and contributes to their acidification.
Its disruption in mice entails a broad defect in renal endocytosis and causes
secondary changes in calciotropic hormone levels. Inactivating mutations in
Dent?s disease lead to proteinuria and kidney stones. Possibly by recycling, a
small fraction of ClC-5 also reaches the plasma membrane. We identified a
carboxy-terminal internalization motif in ClC-5. It resembles the so called PY
motif which is crucial for the endocytosis and degradation of epithelial
Na+-channels (ENaC). Disrupting this motif increases surface expression and
currents about 2-fold. This is likely due to interactions with WW-domains as
dominant negative mutants of the ubiquitin-protein ligase WWP2 increased
surface expression and currents of ClC-5 only when its PY motif was intact.
Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant
decreased WT ClC-5 currents, but did not affect channels with mutated motifs.
Similarly, decreasing endocytosis by expressing the inactive S34N mutant of
rab5 increased ClC-5 currents only if its PY motif was intact. Thus, the
endocytosis of ClC-5, which itself is crucial for the endocytosis of other
proteins, depends on the interaction of a carboxy-terminal internalization
signal with ubiquitin-protein ligases containing WW-domains
Lysosomal Membrane Proteins
Schwake M, Schröder B, Saftig P. Lysosomal Membrane Proteins and Their Central Role in Physiology. Traffic. 2013;14(7):739-748.The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non-mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways
Surface Expression and Single Channel Properties of KCNQ2/KCNQ3, M-type K+ Channels Involved in Epilepsy
Schwake M, Pusch M, Kharkovets T, Jentsch TJ. Surface Expression and Single Channel Properties of KCNQ2/KCNQ3, M-type K+ Channels Involved in Epilepsy. Journal of Biological Chemistry. 2000;275(18):13343-13348.Mutations in either KCNQ2 or KCNQ3 underlie benign familial neonatal convulsions (BFNC), an inherited epilepsy. The corresponding proteins are co-expressed in broad regions of the brain and associate to heteromeric K+ channels. These channels mediate M-type currents that regulate neuronal excitability. We investigated the basis for the increase in currents seen after co-expressing these subunits in Xenopus oocytes. Noise analysis and single channel recordings revealed a conductance of â 18 pS for KCNQ2 and â7 pS for KCNQ3. Different conductance levels (ranging from 8 to 22 pS) were seen upon co-expression. Their weighted average is close to that obtained by noise analysis (16 pS). The open probability of heteromeric channels was not increased significantly. Co-expression of both subunits increased the surface expression of KCNQ2 and KCNQ3 by factors of 5 and >10, respectively. A KCNQ2 mutant associated with BFNC that has a truncated cytoplasmic carboxyl terminus did not reach the surface and failed to stimulate KCNQ3 surface expression. By contrast, several BFNC-associated missense mutations in KCNQ2 or KCNQ3 did not alter their surface expression. Thus, the increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly due to an increase in surface expression, which is dependent on an intact carboxyl terminus
- âŠ