Structural and functional studies on the regulation of the protease-chaperone function of DegP and DegQ from Escherichia coli

Die fehlerhafte Entfernung von ungefalteten Proteinen kann zur Bildung von möglicherweise gefährlichen Aggregaten, zur Inaktivierung von funktionellen Proteinen bis hin zum Tod einer Zelle führen. Um eine einwandfreie Homöostase aufrechtzuerhalten hat die Zelle ein System zur Überwachung der Proteinqualität entwickelt. Dieses Kontrollsystem umfasst spezielle Chaperone und Proteasen, die den Zustand von zellulären Proteinen unter physiologischen und unter Stressbedingungen überwachen. Abhängig vom Grad der Beschädigung der nicht-nativen Proteine werden diese entweder rückgefaltet oder aber entfernt. In Escherichia coli wird der Zustand von periplasmatischen Proteinen von zwei Repräsentanten der HtrA Proteasefamilie überwacht: DegP und DegQ. Das Hitzeschockprotein DegP verfügt über eine abbauende und eine rückfaltende Aktivität und es kann zwischen diesen beiden gegensätzlichen Funktionen in regulierter Weise umschalten. In dieser Arbeit wurden verschiedene DegP/Substrat-Komplexe charakterisiert. Es zeigte sich, dass fehlgefaltete Proteine das hexamere DegP in große, katalytisch aktive 12- und 24-mere Partikel umwandeln, abhängig von der Größe und der Konzentration des vorliegenden Substrates. Die gleiche Art der Regulation, d.h. eine Aktivierung der Proteaseaktivität durch eine substratinduzierte Umwandlung des Oligomers, konnte auch für DegQ festgestellt werden. Diese Beobachtung deutet darauf hin, dass dieser einzigartige Regulationsmechanismus ein konserviertes Merkmal der HtrA Familie darstellt. Weiterhin zeigte die strukturelle und biochemische Analyse von DegP im Komplex mit Außenmembranproteinen (outer membrane proteins, OMPs), dass DegP Proteine in einer zentralen Kammer einschließt, die sowohl als Chaperon als auch Protease-Kompartiment dienen kann. Während die Einkapselung von gefalteten OMP Protomeren schützend wirkt und möglicherweise den sicheren Transport durch das Periplasma gewährleistet, werden fehlgefaltete Proteine in der molekularen Reaktionskammer abgebaut. Um weitere typische Merkmale der HtrA Familie zu bestimmen, konzentrierte sich diese Studie darauf, regulatorische und mechanistische Unterschiede zwischen den beiden eng verwandten Protease-Chaperon Systemen, DegP und DegQ zu ermitteln. Untersuchungen der Proteaseaktivität und des Molekulargewichtes des DegQ Oligomers mittels Gelpermeationschromatografie ergaben, dass niedrige pH-Werte (5.5) eine Umwandlung von DegQ von einem hexameren zu einem möglicherweise dodekameren Zustand induzieren. Auffälligerweise war die Veränderung des oligomeren Zustandes mit einer Veränderung der Proteaseaktivität verbunden, die im Gegensatz zu DegP, am höchsten bei niedrigen pH-Werten war. Bei der weitergehenden Untersuchung der in vivo Relevanz dieser Beobachtung zeigte sich, dass DegQ vor allem für das Wachstum von Escherichia coli bei niedrigen pH-Werten wichtig ist. Dies deutet darauf hin, dass die pH-abhängige Regulation von DegQ die Adaption des Bakteriums an eine Umgebung mit veränderbarem pH-Wert wiederspiegelt. Weiterhin zeigte das Wachstum eines degQ null Stammes eine verlängerte Adaptionsphase im Vergleich zum Wildtyp, was auf eine grundlegende Rolle von DegQ in der Proteinhomöostase hinweist, welche essentiell in der äußerst instabilen Umgebung der bakteriellen Zellhülle ist.The failure to eliminate misfolded proteins can cause the formation of potentially toxic aggregates, inactivation of functional proteins and ultimately cell death. In order to sustain proper homeostasis cells have developed a system of protein quality surveillance. It involves dedicated chaperones and proteases to monitor and control the state of cellular proteins and depending on the degree of damage, either refold or digest aberrant proteins. The pool of periplasmic proteins of E .coli is quality-controlled by two representatives of the HtrA proteases family, namely DegP and DegQ. The heat-shock protein DegP combines digestive and remodelling activities and can switch between these antagonistic functions in a tightly regulated manner. In this study the characterization of different DegP/substrate complexes revealed that binding of misfolded proteins transformed hexameric DegP into large, catalytically active 12- and 24-meric multimers dependent on the size and concentration of the substrate. The same mode of regulation, i.e. protease activation by substrate-induced oligomer reassembly, also appears in DegQ indicating that this unique regulatory mechanism is a conserved feature of HtrA proteins. Moreover, structural and biochemical analysis of DegP complexes with outer membrane proteins (OMPs) revealed that DegP represents a protein packaging device whose central compartment serves antagonistic functions. While encapsulation of folded OMP protomers is protective and might allow safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber. In parallel to elucidate common HtrA features, this study focused on regulatory and mechanistic differences between the two closely related protease-chaperones DegP and DegQ. Activity assays and size exclusion chromatography analysis demonstrated that low pH (5.5) induces remodeling of the DegQ particle, most likely from hexamer to dodecamer. Remarkably, the conversion of the oligomeric state was accompanied by a change in the protease activity being, in contrast to DegP, the most pronounced at low pH. In vivo DegQ was shown to affect the growth of E. coli at lower pH values, while the presence of DegP had no effect. Thus the pH dependent activity of DegQ might reflect adaptation of the bacterium to habitats with variable pH values. Furthermore, the growth of degQ null mutant strain shows an elongated adaptation phase compared to the wild type, indicating an important house keeping function of DegQ, which is essential in the highly unstable environment of the bacterial envelope

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy

UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin‐fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER‐bound ribosomes and activates C53‐mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8‐interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM‐mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation‐dependent fine‐tuning of C53‐mediated autophagy activation

Membrane curvature at a glance

Membrane curvature is an important parameter in defining the morphology of cells, organelles and local membrane subdomains. Transport intermediates have simpler shapes, being either spheres or tubules. The generation and maintenance of curvature is of central importance for maintaining trafficking and cellular functions. It is possible that local shapes in complex membranes could help to define local subregions. In this Cell Science at a Glance article and accompanying poster, we summarize how generating, sensing and maintaining high local membrane curvature is an active process that is mediated and controlled by specialized proteins using general mechanisms: (i) changes in lipid composition and asymmetry, (ii) partitioning of shaped transmembrane domains of integral membrane proteins or protein or domain crowding, (iii) reversible insertion of hydrophobic protein motifs, (iv) nanoscopic scaffolding by oligomerized hydrophilic protein domains and, finally, (v) macroscopic scaffolding by the cytoskeleton with forces generated by polymerization and by molecular motors. We also summarize some of the discoveries about the functions of membrane curvature, where in addition to providing cell or organelle shape, local curvature can affect processes like membrane scission and fusion as well as protein concentration and enzyme activation on membranes

Post‐translationally‐modified structures in the autophagy machinery: an integrative perspective

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113777/1/febs13356_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113777/2/febs13356.pd

Structural properties of the human acidic ribosomal P proteins forming the P1-P2 heterocomplex.

The ribosome has a morphologically distinct structural feature called the stalk, recognized as a vital element for its function. The ribosomal P proteins constitute the main part of the eukaryotic ribosomal stalk, forming a pentameric structure P0-(P1-P2)(2). The group of P1/P2 proteins in eukaryotes is very diverse, and in spite of functional and structural similarities they do not fully complement one another, probably constituting an adaptive feature of the ribosome from a particular species to diverse environmental conditions. The functional differences among the P1/P2 proteins were analysed in vivo several times; however, a thorough molecular characterization was only done for the yeast P1/P2 proteins. Here, we report a biophysical analysis of the human P1 and P2 proteins, applying mass spectrometry, CD and fluorescence spectroscopy, cross-linking and size exclusion chromatography. The human P1/P2 proteins form stable heterodimer, as it is the case for P1/P2 from yeast. However, unlike the yeast complex P1A-P2B, the human P1-P2 dimer showed a three-state transition mechanism, suggesting that an intermediate species may exist in solution

Stress eating: Autophagy targets nuclear pore complexes

Lee et al. (2020. Nat. Cell Biol.https://doi.org/10.1038/s41556-019-0459-2) and, in this issue, Tomioka et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201910063) describe the targeted degradation of nuclear pore complexes (NPCs) by selective autophagy, providing insight into the mechanisms of turnover for individual nucleoporins and entire NPCs

Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum