Das Hsc/Hsp70 Co-Chaperon-Netzwerk kontrolliert die Antigenaggregation und -präsentation während der Immunzellreifung

Während der Reifung von murinen Makrophagen und BMDCs (bone marrow derived dendritic cells) akkumulieren transient ubiquitinierte Proteine in DALIS (Dendritic Cell Aggresome-Like Induced Structures). DALIS beinhalten defekte ribosomale Proteine (DRiPs - defective ribosomal products), die prozessiert und auf MHCI-Molekülen präsentiert werden. Dadurch werden cytotoxische T-Zellen (CTLs - cytotoxic T-lymphocytes) aktiviert und eine Immunantwort eingeleitet. In vorliegender Arbeit wurde am Beispiel der Formation von DALIS untersucht, wie die Aggregat-Bildung in eukaryotische Zellen durch Modulation der Proteostase- Maschinerie beeinflusst wird. Tatsächlich konnte das Chaperon/Co-Chaperon- Netzwerk als Regulator der Bildung und des Abbaus von DALIS identifiziert werden. Co-Chaperone modulieren die Interaktion zwischen Hsc/Hsp70 und dem Ubiquitin/Proteasomsystem, bzw. dem autophagischen Abbau. Die Chaperon-assoziierte Ubiquitinligase CHIP und das Ubiquitin-ähnliche Protein BAG-1 werden essentiell für die Bildung von DALIS in murinen Makrophagen und BMDCs benötigt. DesWeiteren kooperiert CHIP mit dem Autophagie-assoziierten Co-Chaperon BAG-3 und dem Ubiquitin-Adapter p62 und vermittelt den Abbau von DALIS über Chaperon-vermittelte selektive Autophagie (CASA - chaperone-assisted selective autophagy). Das Co-Chaperon HspBP1 inhibiert CHIP-Aktivität und drosselt auf diese Weise die Einschleusung von Peptiden in den Weg der Antigenprozessierung. Durch die Regulation der DALIS-Formation kontrollieren die Co-Chaperone die Präsentation von endogenen und viralen Antigenen auf MHCI-Molekülen in murinen BMDCs. In vorliegender In vorliegender Arbeit konnte zum ersten Mal gezeigt werden, dass das Chaperon/ Co-Chaperon-Netzwerk die transienten Protein-Aggregation in professionellen Antigen-präsentierenden Zellen (APC - antigen presenting cell) kontrolliert und auf dieseWeise die Immunantwort reguliert. Ähnliche Mechanismen könnten auch bei der Formation von Aggresomen oder Aggresomen-ähnlichen Aggregaten in Nicht-Immunzellen eine Rolle spielen

The Hsc/Hsp70 Co-Chaperone Network Controls Antigen Aggregation and Presentation during Maturation of Professional Antigen Presenting Cells

The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS). Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS) and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs). CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA). On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS) in other mammalian cell types

BMDCs isolated from <i>hspBP1</i>^−/− mice show increased DALIS formation during maturation.

<p>(A) BMDCs were isolated from <i>hspBP1</i>^−/− mice and heterozygous siblings and treated with LPS. At indicated time points cell extracts were prepared and analyzed by immune blotting or subjected to the filter trap assay for DALIS detection. Signal intensity for detergent insoluble ubiquitin conjugates was quantified. Value at time point zero for heterozygous cells was set to 1. Error bars represent sem from three independent experiments. (B) DALIS were quantified in BMDCs from <i>hspBP1</i>−/− mice and heterozygous siblings by immune fluorescence with FK2 antibody after LPS treatment for 12 h. Error bars represent sem from three independent experiments.</p

The co-chaperones CHIP, BAG-1, BAG-3, and HspBP1 regulate chaperone-assisted degradation.

<p>Chaperone-assisted degradation is initiated when CHIP binds to the carboxy-terminus of Hsc/Hsp70 (‘C’). After CHIP-mediated recruitment of ubiquitin conjugating enzymes of the Ubc4/5 family, the chaperone-bound protein substrate is modified by attachment of a ubiquitin chain. The co-chaperones BAG-1, BAG-3 and HspBP1 regulate CHIP function. They are able to bind to the amino terminal ATPase domain of Hsc/Hsp70 (‘N’) at the same time when CHIP occupies the carboxy-terminus. Binding of BAG-1 initiates sorting to the proteasome (‘prot.’), whereas BAG-3 triggers the recruitment of the autophagic ubiquitin adaptor p62 and thus facilitates substrate degradation through the autophagosome-lysosome pathway. In contrast, HspBP1 inhibits the ubiquitin ligase activity of CHIP in the formed chaperone complex and thereby abrogates chaperone-assisted degradation. (‘P’ - peptide binding domain of Hsc/Hsp70; ‘auto.’ - autophagosome; ‘lyso.’ - lysosome).</p

CHIP and BAG-1 are essential for DALIS formation, whereas HspBP1 exerts an attenuating function.

<p>(A) Co-chaperones were depleted from RAW309 macrophages by addition of specific siRNAs for 24 h followed by addition of LPS. Control cells received an equal amount of scrambled siRNA. At indicated time points cell extracts were prepared and analyzed by immune blotting or subjected to a filter trap assay for DALIS detection. Signal intensity for detergent insoluble ubiquitin conjugates was quantified. Value at time point zero for control cells was set to 1. Error bars represent sem from three independent experiments. (B) Same as under A, except for the use of mouse BMDCs.</p

Diverse proteostasis components co-localize with antigens in DALIS.

<p>(A) Using specific antibodies against diverse proteostasis components Hsp70, CHIP, BAG-1, BAG-3, and LC3 were detected in DALIS between 12 and 24 h after LPS addition to RAW309 macrophages. In contrast, the co-chaperone HspBP1 did not colocalize with ubiquitin conjugates in DALIS. DALIS were detected with the FK2 antibody that recognizes ubiquitin conjugates (‘ub-conj.’). Scale bars correspond to 5 µm. (B) The amount of DALIS that stained positive for BAG-1, BAG-3 and LC3 were quantified at the indicated time points after LPS addition. Error bars represent sem from three independent experiments. (C) RAW309 macrophages were infected with an ovalbumin expressing adenovirus and stimulated with LPS for 12 h prior to immune fluorescence. Ovalbumin was detected in DALIS with a specific antibody (‘α-OVA’), while ubiquitin conjugates (‘ub-conj.’) were visualized with the FK2 antibody. Scale bar: 5 µm. (D) RAW309 macrophages were infected with Ad-OVA and stimulated with LPS for 12 h. Control cells were left untreated or treated with LPS only. Cells were lysed in detergent containing buffer and 50 µg of total protein were passed over a nitrocellulose membrane. DALIS were detected with FK2 antibody (‘ub-conj.’) and ovalbumin with a specific antibody (‘α-OVA’).</p

DALIS are detectable by immune fluorescence and a filter trap assay.

<p>(A) Following addition of LPS to RAW309 macrophages and mouse BMDCs ubiquitin conjugates were detected by immune fluorescence using the FK2 antibody. Transient DALIS formation peaks around 12 h after LPS addition. Scale bars: 10 µm. (B) Immune cells were lysed at the indicated time points during LPS-induced maturation in detergent containing buffer and passed through a nitrocellulose filter. Detergent insoluble ubiquitin conjugates were detected on the filter with the FK2 antibody. 50 µg of cell lysate were passed through each dot.</p

Over-expression of CHIP and BAG-1 increases DALIS formation, whereas HspBP1 and BAG-3 attenuate formation.

<p>(A) Co-chaperone levels were determined by immune blotting with specific antibodies following transfection of RAW309 macrophages with equal amounts of empty pcDNA3.1 plasmid (‘−’) or the same vector containing the coding region for the indicated co-chaperone (‘+’). Cell lysates were prepared 24 h after transfection. (B) RAW309 macrophages were transfected with empty plasmid (‘control’) or an equal amount of a co-chaperone encoding plasmid for 24 h followed by LPS addition for 12 h. DALIS formation was analyzed using the filter trap assay. Detergent insoluble ubiquitin conjugates (‘ub-conj.’) were detected on the filter with FK2 antibody and signal intensity was quantified. Value for LPS-stimulated control cells was set to 1. Error bars represent sem from at least three independent experiments. (C) Representative immune fluorescence micrographs of transfected macrophages after over-expression of the indicated co-chaperones for 24 h followed by LPS stimulation for 12 h. Lower panel shows a quantification of obtained data. Number of DALIS observed in control cells were set to 1. Error bars represent sem from at least three independent experiments. Scale bars: 10 µm.</p

The Hsc/Hsp70 co-chaperone network controls DALIS formation during immune cell maturation.

<p>Immune cell maturation can be separated in two distinct phases that are characterized by distinct chaperone environments. During early stages of immune cell maturation, characterized by DALIS formation (aggregation phase), Hsc/Hsp70 binds defective ribosomal products (DRiPs) following their translation. Recruitment of CHIP leads to the ubiquitylation of DRiPs, which provides a sorting signal for sequestration into DALIS. Ubiquitylation activity is regulated by the CHIP-inhibitor HspBP1. DALIS formation also requires BAG-1. Late stages of maturation (presentation phase), which involve antigen presentation and clearance of DALIS, are characterized by decreased HspBP1 levels and induction of Hsp70, BAG-1, and BAG-3. These changes stimulate DRiP processing and presentation. In addition, BAG-3 is recruited to DALIS to facilitate aggregate clearance by chaperone-assisted selective autophagy in cooperation with p62. It remains to be seen whether this autophagy pathway contributes to MHC class I mediated presentation (‘?’).</p

Nrf2 activates augmenter of liver regeneration (ALR) via antioxidant response element and links oxidative stress to liver regeneration.

Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2–related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2+/+ mice compared with Nrf2−/− knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells