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

Molecular adaptation of the DegQ protease to exert protein quality control in the bacterial cell envelope

To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of Escherichia coli is monitored by the HtrA proteases DegS, DegP, and DegQ. In contrast with DegP and DegS, the structure and function of DegQ has not been addressed in detail. Here, we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12- and 24-meric HtrA complexes reveals a conserved PDZ1 → L3 → LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope

The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel class of Old Yellow Enzymes

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs

Regulation of the σE stress response by DegS: how the PDZ domain keeps the protease inactive in the resting state and allows integration of different OMP-derived stress signals upon folding stress

The unfolded protein response of Escherichia coli is triggered by the accumulation of unassembled outer membrane proteins (OMPs) in the cellular envelope. The PDZ-protease DegS recognizes these mislocalized OMPs and initiates a proteolytic cascade that ultimately leads to the σE-driven expression of a variety of factors dealing with folding stress in the periplasm and OMP assembly. The general features of how OMPs activate the protease function of DegS have not yet been systematically addressed. Furthermore, it is unknown how the PDZ domain keeps the protease inactive in the resting state, which is of crucial importance for the functioning of the entire σE stress response. Here we show in atomic detail how DegS is able to integrate the information of distinct stress signals that originate from different OMPs containing a ϕ-x-Phe C-terminal motif. A dedicated loop of the protease domain, loop L3, serves as a versatile sensor for allosteric ligands. L3 is capable of interacting differently with ligands but reorients in a conserved manner to activate DegS. Our data also indicate that the PDZ domain directly inhibits protease function in the absence of stress signals by wedging loop L3 in a conformation that ultimately disrupts the proteolytic site. Thus, the PDZ domain and loop L3 of DegS define a novel molecular switch allowing strict regulation of the σE stress response system

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

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

UFMylation mediates the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and regulates the selective degradation of endoplasmic reticulum (ER) via autophagy (ER-phagy) to maintain ER homeostasis. Specifically, collisions of the ER-bound ribosomes trigger ribosome UFMylation, which in turn activates C53-mediated autophagy that clears the toxic incomplete polypeptides. C53 has evolved non-canonical shuffled ATG8 interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown. Here, using a phylogenomics approach, we show that UFMylation is conserved across the eukaryotes and secondarily lost in fungi and some other species. Further biochemical assays have confirmed those results and showed that the unicellular algae, Chlamydomonas reinhardtii has a functional UFMylation machinery, overturning the assumption that this process is linked to multicellularity. Our conservation analysis also revealed that UFM1 co-evolves with the sAIMs in C53, reflecting a functional link between UFM1 and the sAIMs. Using biochemical and structural approaches, we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound to the sAIMs in a different mode. Conversion of sAIMs into canonical AIMs prevented binding of UFM1 to C53, while strengthening ATG8 interaction. This led to the autoactivation of the C53 pathway and sensitized Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral toggle switch embodied in the sAIMs that regulates C53-mediated autophagy to maintain ER homeostasis.Competing Interest StatementThe authors have declared no competing interest