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Institute
Ubiquitin and the ubiquitin-like protein ATG8 are covalently attached to their respective targets via a coordinated cascade involving E1 activating, E2 conjugating and E3 ligating enzymes. Whereas ubiquitin is conferred to proteins as mono- and/or polymer(s) to alter their stability, localization and/or activity, the ubiquitin-like modifier (UBL) ATG8 is conjugated to the phospholipid phosphatidylethanolamine (PE). The best understood function of ATG8 is during autophagy where ATG8-PE conjugates are incorporated into both layers of incipient autophagosomes and serve as multipurpose docking sites for autophagosomal cargo receptors as well as regulatory factors (termed adaptors) that drive formation and maturation of autophagosomes. Mammalian cells harbor six ATG8 family members that can be subclassified into the LC3- and GABARAP-family and that can all be lipidated. However, it is currently unclear to what extent these proteins are functionally redundant or fulfil unique roles.
Cullin-RING ligase complexes (CRLs) are modular E3 ubiquitin ligases that comprise a RING-finger protein that associates with the ubiquitin-charged E2 enzyme, a substrate recruiting module as well as a cullin scaffold as a linker between RING protein and substrate adaptor. Whereas SCF (SKP1-CUL1-F-box protein) complexes, the most studied CRLs, harbor cullin-1 (CUL1) as scaffold and F-box proteins as substrate binding modules, CUL3-containing CRL complexes employ cullin-3 (CUL3), RING-box protein 1 (RBX1) and BTB proteins as substrate adaptors. Here, the BTB domain serves as binding interface for CUL3 and is usually complemented by an additional protein-protein interaction domain such as MATH or Kelch that mediates binding to the substrate of the E3 ligase complex.
Besides ubiquitylation, guanine nucleotide binding is another common way to regulate protein activity and signaling in cells. Here, small Rho GTPases cycle between active and inactive states by binding of the guanine nucleotides GTP or GDP with the help of regulatory proteins. Whereas GTPase-activating proteins (GAP) render RAC1 inactive by facilitating GTP hydrolysis, guanine exchange factors (GEF) such as T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1) activate RAC1 by stimulating the exchange of GDP to GTP. Local control of RAC1 activity is essential to allow a specific cellular response to stimuli such as growth factors or migratory impulses.
This study reports an unexpected link between the GABARAP subfamily of mammalian ATG8 proteins, the ubiquitin proteasome system and RAC1 through the ubiquitylation of the RAC1 GEF TIAM1. The Kelch repeat and BTB domain-containing proteins 6 (KBTBD6) and 7 (KBTBD7) were established as heterodimeric substrate adaptors for CUL3. Interestingly, a thorough proteomic analysis revealed a number of putative substrates but, out of 11 substrate candidates tested, only the RAC1 GEF TIAM1 appeared to be influenced by depletion of CUL3KBTBD6/KBTBD7. Binding studies showed that KBTBD7 binds TIAM1 via the Kelch repeats and that this binding was markedly enhanced when CUL3 activation was abolished upon treatment with the neddylation inhibitor MLN4924. Also, total TIAM1 abundance was increased upon CUL3KBTBD6/KBTBD7 depletion and accumulation of TIAM1 upon proteasome inhibition suggested that TIAM1 is degraded via the proteasome. In vivo ubiquitylation assays and denaturing immunoprecipitations as well as mass spectrometrical analysis confirmed that CUL3KBTBD6/KBTBD7 ubiquitylates TIAM1 at two distinct lysines (K1404 and K1420) close to its C-terminus.
Previously, KBTBD6 and KBTBD7 were found as interactors of several members of the human ATG8 family of proteins in a proteomic study analyzing the human autophagy network. This association was confirmed in the present work. Furthermore, peptide array technology and mutational analysis revealed that KBTBD6 and KBTBD7 employ a classical ATG8-family interacting motif (AIM; also referred to as LC3-interacting region or LIR) as binding interface. The AIMs of KBTBD6 (W-V-R-V) and KBTBD7 (W-V-Q-V) fulfil the consensus AIM sequence motif (F/W/Y1-X2-X3-I/L/V4) and are preceded by several acidic residues and serines. A series of structural and cell biological experiments revealed a binding preference for the GABARAP subfamily of human ATG8 proteins and most importantly, a requirement of the GABARAP-KBTBD6 and -KBTBD7 interaction for TIAM1 ubiquitylation. The finding that TIAM1 binding to KBTBD6 and KBTBD7 AIM mutants was diminished raised the possibility that GABARAP binding mediates the recruitment of CUL3KBTBD6/KBTBD7 to membranes where TIAM1 is localized. Interestingly, colocalization of KBTBD6, GABARAPL1 and TIAM1 in punctuate structures could be observed. Since only a very small fraction of GABARAPL1 colocalized with LC3B, and colocalization between KBTBD6 and LC3B was not observed, these vesicular structures are most likely distinct from autophagosomes. Furthermore, TIAM1 ubiquitylation was reduced when GABARAP, but not LC3B, was depleted or when lipidation of GABARAP was prevented.
Stabilization of TIAM1 upon KBTBD6 and/or KBTBD7 depletion led to elevated TIAM1-dependent RAC1 activity, altered actin morphology with increased cortical actin and loss of vinculin foci. Re-introduction of wild-type KBTBD6 or KBTBD7 but not AIM mutants reverted all these phenotypes. Moreover, depletion of KBTBD6 or KBTBD7 in human breast cancer cells massively increased their invasiveness, whereas TIAM1 knockdown had the opposite outcome. All physiological effects of KBTBD6 and KBTBD7 depletion were inhibited by additional depletion of TIAM1 or RAC1 confirming that the phenotypes observed are indeed mediated by the CUL3KBTBD6/KBTBD7-TIAM1-RAC1 signaling pathway. Intriguingly, KBTBD6 and KBTBD7 were not subject to autophagosomal degradation, thereby establishing a new function for GABARAP proteins beyond autophagosomal degradation in providing a signaling platform for recruitment of the E3 ligase CUL3KBTBD6/KBTBD7 in close proximity to its substrate TIAM1, enabling localized ubiquitylation.
Local restricted control of RAC1 activity by ubiquitylation has been described for TIAM1-RAC1 signaling previously. Examples are HECT, UBA and WWE domain-containing protein 1 (HUWE1)-mediated TIAM1 ubiquitylation that occurs predominantly at cell-cell-junctions in response to hepatocyte growth factor stimulation in MDCKII cells or inhibition of RAC1 activity by the RAC1 GAP protein BCR (breakpoint cluster region) at the leading edge of astrocytes through binding to the TIAM1-Par (polarity) complex. SCFBTRC mediates ubiquitylation of TIAM1 in response to mitogens or DNA damage, though it has not been explored whether this regulation is spatially restricted. Thus, this study adds a novel layer of complexity to the spatial regulation of RAC1 signaling by implicating membrane-bound human ATG8 proteins in this process.
Also, this study is the first report specifically implicating the GABARAP proteins in cellular signaling events. It will be interesting to explore whether the concept of localized signaling mediated by GABARAPs applies to other substrates of CUL3KBTBD6/KBTBD7 and membranerelated signaling processes in which GABARAP proteins are involved. Controlling RAC1 activity at GABARAP-decorated membranes might also be important for trafficking events or autophagy since it was described that RAC1 has an inhibitory function on autophagy. Therefore, spatial restricted ubiquitylation of TIAM1 resulting in specific deactivation of RAC1 could promote the autophagic process when locally needed. Although the catalytic mTOR inhibitor Torin1 and the lysosomal H+ ATPase inhibitor BafilomycinA1 promoted TIAM1 ubiquitylation by increasing the pool of membrane-conjugated GABARAP, but other signals that stimulate GABARAP-KBTBD6/KBTBD7 association and subsequent TIAM1 ubiquitylation are to be identified. Besides, determining the KBTBD6/KBTBD7 binding site in TIAM1 or uncovering a deubiquitylating enzyme (DUB) that locally counteracts the ubiquitylation of TIAM1 will enable a better comprehension of the complete localized signaling cascade.
Tectonin β-propeller containing protein 2 (TECPR2) was first identified in a mass- spectrometric approach as an interactor of GABARAP, an ATG8-family protein playing a role in autophagy. The mammalian ATG8 protein family consists of seven members, namely MAP1LC3A (LC3A), MAP1LC3B (LC3B), MAP1LC3C (LC3C), GABARAP, GABARAPL1 and GABARAPL2. All share an ubiquitin-like core and possess two additional N-terminal α-helices, which are important for the distinct functions of the proteins. First determined in various organelles the ATG8 proteins are shown to be involved in autophagy, supporting the formation and cargo recruitment of autophagosomes, the vesicles transporting cargo for autophagic degradation.
Autophagy is the process of recycling cytoplasmic contents by degradation of misfolded proteins or damaged organelles in order to supply nutrients. Also clearance of pathogens can be achieved via autophagy. Importantly, LC3B is incorporated into the autophagosomal membrane and is therefore used as the main marker for autophagosomes. Previous studies exhibited that depletion of TECPR2 leads to a loss of LC3B-positive structures in cells, which suggests TECPR2 to positively regulate autophagic processes.
A frame shift deletion in the gene encoding for TECPR2 causes the generation of a premature stop codon and subsequent an unstable version of the protein, which is then degraded. Mutation in the TECPR2 gene triggers a neurodegenerative disorder termed hereditary spastic paraparesis (HSP). HSPs are a diverse group of neurodegenerative diseases that are characterized by spasticity in prevalent lower extremities and were mediated by a loss of axonal integrity of the corticospinal motor neurons. In the context of HSP more than 50 gene loci were identified by now. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy causing a form of HSP, the exact function of TECPR2 is unknown.
This study primarily focused on the determination of TECPR2’s binding mode to ATG8 proteins in vitro and in cells. The association of TECPR2 to all ATG8-family proteins was confirmed in in vitro pulldown experiments. Following fragment-based binding and peptide array experiments, the LC3-interacting region (LIR) of TECPR2 could be verified with mutants of TECPR2 lacking the LIR motif. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) were conducted to gain deeper insights into the binding preference to the different ATG8-family members. Moreover, the crystal structure of TECPR2-LIR was solved. In cells colocalization studies with overexpressed ATG8 proteins unraveled a preferential binding to the LC3-subfamily.
Further, mass spectrometric analysis revealed novel association partners of TECPR2: SEC24D, HOPS and BLOC-1, all of those participating in different endomembrane trafficking pathways. Interaction and colocalization of TECPR2 with these components was validated with several immunoprecipitation experiments and the N-terminal part of the protein comprising the WD40-domain could be defined as the binding site for all three of the association partners. In further approaches, the requirement of the LIR-motif and the necessity of the availability of LC3 protein for the particular interactions were determined. Interestingly, in the absence of LC3C the binding of TECPR2 to SEC24D was completely disrupted whereas a loss of LC3B only resulted in a decreased association. Notably, the binding proteins were not subjected to autophagosomal degradation, indicating that TECPR2 may operate as a multifunctional scaffold protein. While depletion of TECPR2 destabilized HOPS and BLOC-1, the autophagy defect observed in TECRP2-deficient cells could not be attributed to functional impairment of these two complexes.
Moreover, loss of TECPR2 led to a decline in protein levels of SEC24D and of its heterodimer partner SEC23A. Thus, TECPR2 is required to regulate the protein levels of SEC23A and SEC24D and subsequently the formation of the heterodimers. Together, SEC24D and SEC23A form the inner coat of COPII vesicles. These vesicles are responsible for the anterograde transport of cargo from the ER toward the Golgi compartment. COPII-coated vesicles are secreted form ER at distinct sites, termed ER exit sites (ERES). The small GTPase SAR1A maintains the vesicle budding, coating and secretion at the ERES. Together with SEC13, SEC31 forms the outer coat of the COPII vesicles and therefore serves as a general ERES marker.
Consistent with a defect in COPII coat assembly, the number of ERES diminished in the absence of TECPR2. These phenotypes could be rescued by the wildtype TECPR2 protein but not by the LIR-mutant. Intriguingly, these results were mimicked by depletion of LC3C, which localized to ERES. By monitoring the release of various cargos from ER in dependency of TECPR2 or LC3C, a role of both proteins in ER export was determined. These facts indicated that TECPR2 cooperates with LC3C to facilitate COPII assembly, ERES maintenance and ER export. Notably, fibroblast derived from a HSP patient carrying mutated TECPR2 showed diminished SEC24D protein levels and delayed ER export.
Concurrent with emerging evidence for a role of ERES in autophagosome formation, depletion of TECPR2 or LC3C or overexpression of a constitutive inactive SAR1 mutant reduced puncta formation of the early autophagosomal protein WIPI2.
In summary, this study uncovered a role for TECPR2 in ER export at ERES through interaction and stabilization of SEC24D, a COPII coat protein. This process also depended on ATG8-family protein LC3C, which is localized at ERES. Both proteins are required for correct COPII-mediated secretion. Moreover, the presence of TECPR2 and LC3C on ER allows development of omegasomes, membranous structures budding ER to form autophagosomes, by stabilization of WIPI2 and therefore contribute to autophagosome formation.
Der Pilz Podospora anserina ist seit mehr als fünf Jahrzehnten ein wichtiger Modellorganismus für die Alternsforschung. Insbesondere die Mitochondrien, essentielle eukaryotische Zellorganellen – wegen ihrer Funktion im Energiestoffwechsel häufig auch als „zelluläre Kraftwerke“ bezeichnet, sind Schlüsselfaktoren für den Alterungsprozess dieses Organismus.
Im Rahmen einer vorangegangenen Diplomarbeit wurde daher der Einfluss der mitochondrialen CLPXP-Protease, einem bisher noch wenig erforschten Bestandteil der Proteinqualitätskontrolle in Mitochondrien, auf die Alterung von P. anserina untersucht. Mitochondriale CLPXP-Proteasen sind, wie auch ihre bakteriellen Pendants, aus zwei verschiedenen Untereinheiten aufgebaut: der Protease-Komponente CLPP und der Chaperon-Komponente CLPX. Die Deletion des Gens PaClpP, kodierend für CLPP in P. anserina, führte zu einer überraschenden Verlängerung der gesunden Lebensspanne der Mutante. Darüber hinaus war es möglich, den pilzlichen PaClpP-Deletionsstamm durch Einbringen von CLPP des Menschen zu komplementieren. Dies beweist, dass die Proteasen CLPP des Menschen und von P. anserina funktionell homolog sind. Dadurch eröffnete sich die Perspektive, diesen einfachen Modellorganismus für die Gewinnung potenziell auf den Menschen übertragbarer Erkenntnisse einzusetzen. Bedeutenderweise ist die menschliche CLPXP-Protease wahrscheinlich involviert in die Entstehung verschiedener Krankheiten, darunter das Perrault-Syndrom sowie einige Krebsarten. Die zugrundeliegenden Mechanismen sind jedoch noch weitestgehend unverstanden.
Ziel des in dieser Dissertation beschriebenen Forschungsprojektes war daher die Gewinnung genauerer Einsichten in die molekulare Funktion und die daraus folgende biologische Rolle der mitochondrialen CLPXP-Protease von P. anserina. Der wohl wichtigste Punkt für das detaillierte Verständnis einer Protease ist die Kenntnis ihres Substratspektrums, d. h. der von ihr abgebauten Proteine. Tatsächlich wurde aber bis heute noch in keinem eukaryotischen Organismus eine umfassende Analyse der Substrate einer mitochondrialen CLPXP-Protease vorgenommen. Um diese Wissenslücke zu füllen, wurde in der vorliegenden Arbeit eine ursprünglich in Bakterien entwickelte Verfahrensweise, der sogenannte CLPP „Substrat-trapping Assay“, in P. anserina implementiert. Dafür mussten zunächst die notwendigen handwerklichen Voraussetzungen für den Assay geschaffen werden, insbesondere die effiziente Affinitätsaufreinigung von Proteinen aus isolierten Mitochondrien – einer bisher in P. anserina noch nicht angewandten Technik. Unter Verwendung verschiedener neu hergestellter Varianten der menschlichen Protease-Komponente CLPP, darunter einer proteolytisch inaktiven Variante zum „Einfangen“ von Substraten, konnte der CLPP „Substrat-trapping Assay“ in P. anserina erfolgreich durchgeführt werden. Insgesamt wurden, in Kooperation mit der Arbeitsgruppe von Julian D. Langer (Max-Planck-Institut für Biophysik; Durchführung von massenspektrometrischen Analysen) nahezu 70 spezifische Proteine erstmalig als potenzielle Substrate oder Interaktionspartner einer mitochondrialen CLPXP-Protease identifiziert. Bei einem Großteil dieser Proteine handelt es sich um Enzyme und Komponenten verschiedener Stoffwechselwege – vor allem um solche, die eine zentrale Rolle im mitochondrialen Energiestoffwechsel spielen. Die Ergebnisse der vorliegenden Arbeit legen somit folgende Arbeitsthese als Schlussfazit und gleichzeitig Ausganspunkt für zukünftige Untersuchungen nahe:
Die hauptsächliche molekulare Funktion der mitochondrialen CLPXP-Protease in P. anserina ist die Degradation von Stoffwechselenzymen und ihre biologische Rolle demnach die Kontrolle und Aufrechterhaltung des mitochondrialen und zellulären Energiestoffwechsels.
Insgesamt ist die auf Grundlage des CLPP „Substrat-trapping Assay“ in P. anserina anzunehmende Rolle der mitochondrialen CLPXP-Protease als regulatorische Komponente des mitochondrialen Energiestoffwechsels erstaunlich gut mit Beobachtungen in anderen eukaryotischen Organismen, gerade bezüglich der Relevanz der CLPXP-Protease des Menschen für diverse Krankheiten, zu vereinbaren. Somit erscheint es überaus sinnvoll und vielversprechend, dass in dieser Doktorarbeit erstellte und bisher beispiellose Kompendium potenzieller in vivo Substrate und Interaktionspartner dieser Protease auch als Referenz für zukünftige Untersuchungen außerhalb von P. anserina anzuwenden.
