Die Rolle des ubiquitin-ähnlichen Proteins FAT10 in Proteinabbau und Immunität

FAT10 ist ein Ubiquitin-ähnliches Protein, welches im Haupt-Gewebe-Kompatibilitäts (MHC) Lokus der Klasse I kodiert liegt. Mit den proinflammatorischen Zytokinen TNF-alpha und IFN-gamma ist es in Zellen fast jeder Herkunft induzierbar. FAT10 besteht aus zwei durch einen kurzen Linker verbundenen Ubiquitin-ähnlichen Domänen und besitzt an seinem C-terminus ein Di-Glycin Motiv, über welches es an seine Zielproteine kovalent konjugiert werden kann. Überexpression des wild-typ Proteins - aber nicht einer Mutante ohne Di-GlycinMotiv - führt zur Induktion von Caspase-abhängiger Apoptose in murinen und humanen Zelllinien. Sowohl FAT10 als auch seine Konjugate werden vom Proteasom mit der gleichen Geschwindigkeit abgebaut; und die N-terminale Fusion von FAT10 an langlebige Proteine wie GFP oder DHFR reduziert deren Halbwertszeit ebenso potent wie eine Fusion mit Ubiquitin. Der proteasomale Abbau von FAT10 - welcher unabhängig von Ubiquitin erfolgt - kann zusätzlich beschleunigt werden durch nicht-kovalente Interaktion von FAT10 mit dem UBL-UBA Protein NEDD8 Ultimate Buster 1-Long (NUB1L).Die vorliegende Arbeit untersucht die molekularen Faktoren dieser Beschleunigung und deckt eine doppelte Rolle für NUB1L sowohl als proteasomaler Rezeptor für FAT10 als auch als sogenannter "Facilitator" seines Abbaus auf. Obwohl FAT10 in der Lage ist, direkt mit dem 26S Proteasom zu interagieren, kann NUB1L als löslicher FAT10-Rezeptor fungieren indem es über seine UBL Domäne an das Proteasom und über seine drei UBA Domänen an FAT10 bindet. Interessanterweise ist die Interaktion zwischen NUB1L und FAT10 - zumindest in vivo - abhängig vom Vorhandensein aller drei UBA Domänen. Im Gegensatz dazu beruht Beschleunigung des FAT10-Abbaus alleine auf der Fähigkeit von NUB11L, über seine UBL Domäne an das 26S Proteasom zu binden. Des weiteren kann der Abbau der N-terminalen UBL Domäne von FAT10 - welche mit dem 26S Proteasom, aber nicht mit NUB1L interagieren kann - immer noch durch die Co-Expression von NUB1L beschleunigt werden.Mit Hilfe von Experimenten in einem in vitro System, welches aus aufgereinigtem 26S Proteasom sowie rekombinant exprimierten Substraten bestand, zeigt diese Arbeit, dass das 26S Proteasom durchaus in der Lage ist das Modell-Substrat FAT10-DHFR in vitro abzubauen - allerdings nur in der Gegenwart von NUB1L. Versuche in NUB1L knock-down Zellen lassen den Schluss zu, dass diese absolute Abhängigkeit des FAT10-gesteuerten Abbaus von NUB1L auch auf die Situation in vivo zutrifft. Dies ist ein Hinweis darauf, dass - zumindest im Falle des hier untersuchten Modell-Substrats - die Bindung von FAT10 an das Proteasom alleine nicht ausreicht, um einen effizienten Abbau seiner Zielproteine zu ermöglichen.Neben NUB1L wurde Histon Deacetylase 6 (HDAC6) als weiterer, nicht-kovalenter Interaktionspartner von FAT10 identifiziert. Zusätzlich zu seinen beiden katalytischen Domänen besitzt es eine Dynein-Bindedomäne sowie einen Ubiquitin-bindenden Zink-Finger (BUZ Domäne). Mit Hilfe dieser beiden Domänen fungiert es als Verbindung zwischen dem Dynein-Motor sowie polyubiquitylierten Proteinen und ermöglicht auf diese Weise deren Transport entlang von Mikrotubuli zum Aggresom. Diese Arbeit zeigt, dass HDAC6 nicht nur für den Transport von polyubiquitylierten Proteinen, sondern auch für den Transport von FAT10 und seinen Konjugaten zuständig ist. Unter Bedingungen in denen das Proteasom nur eingeschränkt funktioniert bindet HDAC6 an FAT10 und vermittelt seinen Transport zum Aggresom. Die Interaktion mit FAT10 wird durch zwei verschiedene Domänen von HDAC6 vermittelt, nämlich die BUZ Domäne und die erste katalytische Domäne, obwohl die Interaktion der beiden Proteine nicht von der katalytischen Aktivität von HDAC6 abhängig ist. Des weiteren zeigt diese Arbeit, dass sowohl die Anzahl als auch die Größe von FAT10 und Polyubiquitinhaltigen Aggresomen in HDAC6-defizienten Zellen stark reduziert sind.Zusammengenommen weisen diese Ergebnisse darauf hin, dass FAT10 den schnellen und induzierbaren Abbau seiner Zielprotein durch nicht-kovalente Interaktion mit zwei verschiedenen Adapterproteinen vermittelt. Mit Hilfe des UBL-UBA Proteins NUB1L ist FAT10 in der Lage, seine Zielproteine dem Abbau durch das 26S Proteasom zuzuführen. Ist das Proteasom jedoch beeinträchtigt, vermittelt FAT10 stattdessen über seine Bindung an HDAC6 die Isolierung seiner Zielproteine im Aggresom

The ubiquitin-like modifier FAT10 interacts with HDAC6 and localizes to aggresomes under proteasome inhibition

During misfolded-protein stress, the cytoplasmic protein histone deacetylase 6 (HDAC6) functions as a linker between the dynein motor and polyubiquitin to mediate the transport of polyubiquitylated cargo to the aggresome. Here, we identify a new binding partner of HDAC6, the ubiquitin-like modifier FAT10 (also known as UBD), which is cytokine-inducible and - similar to ubiquitin - serves as a signal for proteasomal degradation. In vivo, the two proteins only interacted under conditions of proteasome impairment. The binding of HDAC6 to FAT10 was mediated by two separate domains: the C-terminal ubiquitin-binding zinc-finger (BUZ domain) of HDAC6 and its first catalytic domain, even though catalytic activity of HDAC6 was not required for this interaction. Both endogenous and ectopically expressed FAT10 as well as the model conjugate FAT10-GFP localized to the aggresome in a microtubule-dependent manner. Furthermore, FAT10-containing as well as ubiquitin-containing aggresomes were reduced in both size and number in HDAC6-deficient fibroblasts. We conclude that, if FAT10 fails to subject its target proteins to proteasomal degradation, an alternative route is taken to ensure their sequestration and possibly also their subsequent removal by transporting them to the aggresome via the association with HDAC6

Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L

The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome, a process accelerated by the UBL-UBA domain protein NEDD8 ultimate buster 1-long. Here, we show that FAT10-mediated degradation occurs independently of poly-ubiquitylation as purified 26S proteasome readily degraded FAT10-dihydrofolate reductase (DHFR) but not ubiquitin-DHFR in vitro. Interestingly, the 26S proteasome could only degrade FAT10-DHFR when NUB1L was present. Knock-down of NUB1L attenuated the degradation of FAT10-DHFR in intact cells suggesting that NUB1L determines the degradation rate of FAT10-linked proteins. In conclusion, our data establish FAT10 as a ubiquitin-independent but NUB1L-dependent targeting signal for proteasomal degradation

Characterization of NSs encoding alanine substitutions at the C-terminus.

<p>(A) Schematics of NSs mutations. Acidic residues, Glu (E) at aa.263–255 and Asp (D) at aa.257–259, were substituted with alanine, and recombinant MP-12 encoding NSs mutations were recovered (NSs-E253-255A/D257–259, NSs-D257-259A and NSs-E253-255A). Underline indicates the amino acids substituted. (B) VeroE6 cells were mock-infected or infected with indicated viruses at an moi of 3. Cells were collected at 16 hpi, and the abundance of PKR (anti-PKR antibody), NSs and N (anti-RVFV antibody), and β-actin (anti-actin antibody) were analyzed by Western blot. (B) A549 cells were mock-infected or infected with indicated viruses at an moi of 3. Total RNA was extracted at 7 hpi, and IFN-β mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe. Representative data from at least 3 independent experiments are shown.</p

Functional Analysis of Rift Valley Fever Virus NSs Encoding a Partial Truncation

<div><p>Rift Valley fever virus (RVFV), belongs to genus <em>Phlebovirus</em> of the family <em>Bunyaviridae</em>, causes high rates of abortion and fetal malformation in infected ruminants as well as causing neurological disorders, blindness, or lethal hemorrhagic fever in humans. RVFV is classified as a category A priority pathogen and a select agent in the U.S., and currently there are no therapeutics available for RVF patients. NSs protein, a major virulence factor of RVFV, inhibits host transcription including interferon (IFN)-β mRNA synthesis and promotes degradation of dsRNA-dependent protein kinase (PKR). NSs self-associates at the C-terminus 17 aa., while NSs at aa.210–230 binds to Sin3A-associated protein (SAP30) to inhibit the activation of IFN-β promoter. Thus, we hypothesize that NSs function(s) can be abolished by truncation of specific domains, and co-expression of nonfunctional NSs with intact NSs will result in the attenuation of NSs function by dominant-negative effect. Unexpectedly, we found that RVFV NSs truncated at aa. 6–30, 31–55, 56–80, 81–105, 106–130, 131–155, 156–180, 181–205, 206–230, 231–248 or 249–265 lack functions of IFN–β mRNA synthesis inhibition and degradation of PKR. Truncated NSs were less stable in infected cells, while nuclear localization was inhibited in NSs lacking either of aa.81–105, 106–130, 131–155, 156–180, 181–205, 206–230 or 231–248. Furthermore, none of truncated NSs had exhibited significant dominant-negative functions for NSs-mediated IFN-β suppression or PKR degradation upon co-expression in cells infected with RVFV. We also found that any of truncated NSs except for intact NSs does not interact with RVFV NSs even in the presence of intact C-terminus self-association domain. Our results suggest that conformational integrity of NSs is important for the stability, cellular localization and biological functions of RVFV NSs, and the co-expression of truncated NSs does not exhibit dominant-negative phenotype.</p> </div

Co-expression of truncated NSs in RVFV-infected cells.

<p>293 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT (control) or NSs with indicated truncations. Cells were collected at 16 hpi, and PKR (anti-PKR antibody), NSs (anti-RVFV antibody), NSs-Flag (anti-Flag antibody), CAT-myc (anti-myc antibody) and β-actin (anti-actin antibody) were detected by Western blot. (B) A549 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs with indicated truncations. Total RNA was extracted at 7 hpi, and IFN-β mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe. Representative data from at least 3 independent experiments are shown.</p

NSs expression of MP-12 and NSΔ249

<p>–<b>265.</b> 293 cells were mock-transfected (mock) or transfected with in vitro transcribed RNA encoding CAT (control) or NSs of MP-12 or NSΔ249–265. At 16 hpi, cells were fixed with methanol for 5 min, and stained with anti-RVFV antibody (1:500) at 37^°C for 1 hr and Alexa Fluor 594, goat anti-mouse IgG (H+L) at 37^°C for 1 hr. After washing with PBS, cells were stained with DAPI, and observed under fluorescent microscope.</p

FAT10, a Ubiquitin-Independent Signal for Proteasomal Degradation

FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitin-like domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-lived proteins enhanced their degradation rate as potently as fusion with ubiquitin did. FAT10-green fluorescent protein fusion proteins were not cleaved but entirely degraded, suggesting that FAT10-specific deconjugating enzymes were not present in the analyzed cell lines. Interestingly, the prevention of ubiquitylation of FAT10 by mutation of all lysines or by expression in ubiquitylation-deficient cells did not affect FAT10 degradation. Thus, conjugation with FAT10 is an alternative and ubiquitin-independent targeting mechanism for degradation by the proteasome, which, in contrast to polyubiquitylation, is cytokine inducible and irreversible

Association of MP-12 NSs and truncated NSs.

<p>293 cells were mock-infected or infected with rMP12-NSs-SF at an moi of 3, and cells were immediately transfected with in vitro synthesized RNA encoding MP-12 NSs, CAT, or truncated NSs. After incubation for 6 hours, newly synthesized proteins were then labeled with [³⁵S] methionine/cycteine. Using cell lysates, SF-tagged proteins were precipitated with Strep-Tactin beads. Then, co-precipitated proteins were analyzed by separating on 10% SDS-PAGE gel and followed by autoradiography. Part of lysates before precipitation were used for Western blot using anti-NSs antibody (input control).</p