Mammalian m-AAA Proteases as Key Regulators of Mitochondrial Function - Analysis of Dominant Negative Mutant Variants

To ensure the removal of excess and non-assembled proteins, mitochondria require a protein quality control system which is constituted by several proteases located in different compartments of mitochondria. m-AAA proteases, oligomeric ATP-dependent metallopeptidases, are key components of this system active at the matrix side of the inner membrane. Human m-AAA proteases build up homo- and hetero-oligomeric complexes composed of AFG3L2 and SPG7. Mice express a third subunit, Afg3l1, resulting in a variety of possible isoenzymes. Interestingly, mutations or deletions of one subunit of mammalian m-AAA proteases cause neurodegeneration in distinct regions of the central and peripheral nervous system in mouse and human, indicating that different tissues, in particular neurons, require a specific subset of isoenzymes. The yeast m-AAA protease can also act as a processing enzyme regulating mitochondrial biogenesis, raising the question which activity is linked to the pathogenesis of the associated diseases. Which molecular functions of mammalian m-AAA proteases contribute to different disease states are poorly understood. Mammalian m AAA proteases have been linked to the processing of the dynamin-like GTPase OPA1 implying a role of mammalian m AAA proteases in mitochondrial fusion. Therefore, to further elucidate the function of mammalian m AAA proteases it was necessary to identify more substrates of the proteases. In this study, a mutation in the Walker B motif of the ATPase domain of Afg3l2/AFG3L2 was identified as dominant negative substrate trap. Using this novel approach, expression of dominant negative mutant variants in human cells, interacting partners and putative substrates have been identified providing further insights into the molecular functions of mammalian m-AAA proteases in mitochondria. These proteases were demonstrated to be present in a supercomplex with prohibitins together regulating cell proliferation and mitochondrial fusion by stabilizing l OPA1. In parallel, m-AAA proteases interact with SLP2 and control stress induced mitochondrial hyperfusion pointing to the formation of another supercomplex containing the proteases and SLP2. The precursor of AFG3L2 itself and MICS1, an inner membrane protein crucial for cristae organization and apoptosis, were identified as possible substrates. Linking m AAA protease functions to mitochondrial fusion, cristae organization and apoptosis may help to unravel the molecular mechanisms underlying neurodegeneration associated with mutations in human m-AAA proteases

Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1

m-AAA proteases cleave OPA1 to ensure a balance of long and short OPA1 isoforms, whereas cleavage by OMA1 causes an accumulation of the short OPA1 variants. (See also companion paper from Head et al. in this issue.

A Few Immobilized Thrombins Are Sufficient for Platelet Spreading

Eukaryotic cells respond to signaling molecules with picomolar to nanomolar sensitivities. However, molar concentrations give no suggestion of the sufficient number of molecules per cell and are confusing when referring to physiological situations in which signaling molecules act in an immobilized state. Here, we studied platelet adhesion by thrombin, a key step in normal hemostasis and pathological arterial thrombosis. We generated a biofunctional nanosheet surface to mimic the in vivo solid-state interaction between platelets and thrombin at sites of injured tissues. We observed that <10 molecules readily activate platelets with high specificity, resulting in platelet adhesion and spreading. This number is much lower than expected from previous experiments in solution, in which the sole activation of platelets required a >1000-fold stoichiometric excess of thrombin. We conclude that immobilizing thrombin apposed to the membrane receptor allows platelets to respond with very high sensitivity. Moreover, we propose that irreversible cell activation may require several ligands to avoid activation by single, mislocalized signaling molecules

SLP-2 is required for stress-induced mitochondrial hyperfusion

Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin-related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress-induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L-OPA1, MFN1, and the mitochondrial inner membrane protein SLP-2. In the absence of SLP-2, L-OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro-survival response against stress

Der Kleinsatellit BIROS in der FireBIRD Mission

Dieser Bericht enthält eine detaillierte Abhandlung des gesamten Entwicklungsprozesses des Bi-spektralen Infrarot-Optischen Systems (BIROS) in der FireBIRD Mission, beginnend mit der wissenschaftlichen Aufgabenstellung zur Detektion und Bewertung von Hochtemperaturereignissen (HTE) aus dem Weltraum über die Auslegung des IR-Kamerasystems als primäre Nutzlast von BIROS, seiner Sekundärnutzlasten, des BIROS Satellitenbusses, dem Nutzerinterface zur Datenanforderung bis hin zu ausgewählten Anwendungsbeispielen der FireBIRD Datenprodukte. Es wird neben der technischen Beschreibung der Subsysteme des Satelliten und der bi-spektralen IR-Kamera, mit Bändern im mittleren Infrarot (MIR) und im thermalenInfrarot (TIR) die adaptive Anpassung der radiometrischen Dynamik der IR-Signaltrakte erklärt. Diese stellt ein Alleinstellungsmerkmal dar im Hinblick auf die bildhafte Erkennung und Bewertung von Feuern oder heißer Lava, welche Temperaturen zwischen 300 °C und 1300 °C erreichen, im sogenannten Sub-Pixelbereich. Anhand von verschiedenen Anwendungsbeispielen wird aufgezeigt, dass mit der IR-Kamera kleine Feuer von nur 10 m2 Ausdehnung zu erkennen sind und gleichzeitig bei der Beobachtung von riesigen Busch-bränden oder groß- flächigen Lavaströmen die IR-Kamera Signaltrakte nicht 'in die Sättigung' gehen, d.h. das Feuersignal nicht begrenzen. Aus der Beobachtung HTE einerseits und von NormalTemperatur-Phänomenen (NTP) konnten die adaptiven Dynamikbereiche für die MIR- und TIRBänder der Kamera nachgwiesen werden, die von keinem anderen IR-Kamaerasystem eines Kleinsatelliten bekannt sind. Die mit BIROS gesammelten Erfahrungen erlauben Schlussfolgerungen für zukünftige Kleinsatellitenmissionen zur räumlich und radiometrisch höher auflösenden Erdbeobachtung im MIR und TIR