PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus

Caractérisation biochimique, structurale et fonctionnelle de PIP30, un nouveau régulateur de l’activateur du protéasome PA28gamma

Le protéasome est responsable de la dégradation régulée d'une majeure partie des protéines intracellulaires. Cette machinerie multimoléculaire est composée d'un cœur catalytique, le protéasome 20S, qui peut être activé par plusieurs types de protéines régulatrices, en particulier la particule régulatrice 19S ou PA700, les complexes heptamériques formés par les membres de la famille 11S (ou PA28) et PA200. Au cours de ce travail, nous nous sommes focalisés sur PA28gamma, un régulateur nucléaire du protéasome, qui active la dégradation de plusieurs substrats par le protéasome 20S de façon indépendante de l'ubiquitine et de l'ATP. Malgré de multiples études montrant l'implication de PA28gamma dans de nombreux processus cellulaires essentiels tels que le cycle cellulaire, la prolifération, l'apoptose, l'architecture nucléaire, la dynamique de la chromatine, les infections virales et la réponse au stress, ses fonctions exactes ne sont pas encore comprises. De plus, les mécanismes impliqués dans la régulation de l'activité de PA28gamma et de son association avec le protéasome 20S restent mystérieux. Une analyse SILAC des partenaires d'interaction de PA28gamma endogène a révélé l'existence d'un nouveau facteur, non caractérisé, que nous avons appelé PIP30 (PA28gamma Interacting Protein 30 kDa). Le gène PIP30 contient un domaine très conservé chez les Eucaryotes. Nous avons produit et purifié la protéine PIP30 recombinante et montré qu'elle est faiblement structurée, malgré le fait qu'elle puisse se dimériser. Nous avons confirmé, aussi bien in vitro qu'in cellulo, que PIP30 interagit directement et spécifiquement avec PA28gamma. En analysant la co-immunoprécipitation de PA28gamma avec différents mutants tronqués de GFP-PIP30, nous avons pu identifier la séquence de PIP30 responsable de l'interaction avec PA28gamma dans sa partie C-terminale. Nous essayons maintenant d'identifier la séquence de PA28gamma impliquée dans la liaison de PIP30 et de cristalliser le complexe PA28gamma/PIP30. L'élaboration d'un anticorps anti-PIP30 « maison » nous a permis de montrer que PIP30 est une protéine nucléaire stable. Son niveau d'expression diminue en réponse à la déplétion de PA28gamma, ce qui suggère que PIP30 est stabilisée par son interaction avec PA28gamma in cellulo. Nous avons démontré in vitro que PIP30 inhibe partiellement l'activation médiée par PA28gamma des activités de type chymotrypsine et caspase, mais pas trypsine, du protéasome. Cependant, nous avons montré, par une approche ELISA, que PIP30 n'affecte pas la liaison de PA28gamma au protéasome 20S. Par ailleurs, nous avons testé l'effet de PIP30 sur la dégradation de p21 par le complexe PA28gamma/protéasome 20S et observé que PIP30 augmente la vitesse de dégradation de p21 dans ce test. Nos tentatives pour élucider la fonction exacte de PIP30 in cellulo n'ont jusqu'ici pas abouti à une conclusion convaincante. L'ensemble de ces résultats suggère que PIP30 pourrait être impliqué dans le recrutement sélectif des substrats de PA28gamma et/ou dans la modulation de l'activation du protéasome par PA28gamma.The proteasome is responsible for the regulated degradation of most intracellular proteins. This multi-subunit machinery is composed of a common catalytic core, the 20S proteasome, which can be activated by various types of regulators, notably the 19S regulatory particle or PA700, the heptameric complexes formed by the members of the 11S (or PA28) family and PA200. This work has been focused on PA28gamma, a nuclear regulator of the proteasome, which has been shown to activate degradation of several proteasomal substrates in an ATP- and ubiquitin- independent manner. Despite many evidences revealing the involvement of PA28gamma in many essential cellular processes, such as cell cycle progression, proliferation, apoptosis, nuclear architecture, chromatin dynamics, viral infection and stress response, its exact function(s) remain to be understood. In addition, how PA28gamma activity and association to the 20S proteasome are regulated is completely unclear. A SILAC-based analysis of endogenous PA28gamma interaction partners revealed the existence of a novel, completely uncharacterized protein, which we called PIP30 (PA28gamma Interacting Protein 30 kDa). Evolutionary analysis indicates that PIP30 gene contains a domain highly conserved in Eukaryotes, without any alternative splicing or gene duplication evidences. We produced and purified the recombinant PIP30 protein and showed that it is poorly structured, although it is able to make dimers. We confirmed both in vitro and in cellulo that PIP30 directly and specifically interacts with PA28gamma. By analyzing the co-immunoprecipitation of PA28gamma with various GFP-PIP30 truncation mutants, we identified the sequence of PIP30 responsible for PA28gamma binding in its C-terminal part. Ongoing analyses now focus on the identification of PIP30 binding motif on PA28gamma sequence and the crystallization of the PA28gamma-PIP30 complex. Using homemade anti-PIP30 antibodies, we showed that PIP30 is a stable nuclear protein. Its expression level is decreased in response to PA28gamma depletion, suggesting that it is stabilized by its interaction with PA28gamma in cellulo. We demonstrated in vitro that PIP30 partially inhibits PA28gamma-mediated activation of the chymotrypsin- and caspase-, but not the trypsin-like, activities of the proteasome. However, we showed by an ELISA-based approach that PIP30 does not affect PA28gamma binding to 20S. Considering the limitations of probing proteasome activity with small fluorogenic substrates, we tested the effect of PIP30 on the PA28gamma-dependent proteasomal degradation of in vitro translated p21, a known protein substrate of PA28gamma. We unexpectedly found that PIP30 enhanced the rate of p21 degradation. Our attempts to elucidate the exact functions of PIP30 in cellulo were unsuccessful so far. Altogether, our results suggest that PIP30 could be involved in the selective recruitment of PA28gamma protein substrates and/or modulate PA28gamma-mediated proteasome activation

The influence of peptidyl arginine deiminases on the biological properties of human cathelicidine

Jednym z potranslacyjnych sposobów modyfikacji białek jest deiminacja reszt argininowych. W wyniku tej reakcji, przeprowadzanej przez enzymy należące do rodziny deiminaz peptydyloarginylowych, eksponowane ugrupowanie guanidynowe reszty argininowej występujące w łańcuchu polipeptydowym ulega deiminacji, w wyniku czego uwalniany jest wolny amoniak, zaś w strukturze polipeptydu pozostaje reszta cytrulinowa. Deiminacja stanowi integralną część mechanizmów zachodzących w organizmie ludzkim podczas procesu rogowacenia naskórka czy też wytwarzania włosa. Jednak, gdy cytrulinacji poddane zostają niefizjologiczne substraty deiminaz peptydyloarginylowych, produkty reakcji zawierające reszty cytrulinowe w sekwencji mogą zostać rozpoznane jako obce antygeny, co przyczynić się może do rozwoju chorób autoimmunologicznych, m.in. reumatoidalnego zapalenia stawów. Wykazano, iż u osób ze zdiagnozowanym reumatoidalnym zapaleniem stawów poziom deiminazy peptydyloarginylowej typu 2 oraz typu 4 jest podwyższony w zmienionych chorobowo stawach. Powiązanie pomiędzy parodontozą i reumatoidalnym zapaleniem stawów zostało skojarzone z obecnością deiminazy peptydyloarginylowej Porphyromonas gingivalis. Poddanie reakcji deiminacji peptydów antybakteryjnych, może mieć wpływ na ich funkcje, ponieważ w wyniku tej reakcji enzymatycznej zmienia się ogólny ładunek cząsteczki. Niniejsza praca udowadnia, iż istotnie katelicydyna LL-37, podobnie jak i mysi peptyd CRAMP, a także krótka syntetyczna forma LL-37(17-37) tracą właściwości bakteriobójcze w wyniku inkubacji z deiminazą peptydyloarginylową pochodzenia króliczego jak i rekombinowaną ludzką deiminazą peptydyloarginylową 2. Dodatkowo, zdolność LL-37 do wiązania endotoksyny LPS również zostaje utracona w wyniku cytrulinacji peptydu.One of post-translational ways to modify proteins is deimination of arginine residues. As the result of this reaction catalyzed by enzymes belonging to the peptidyl arginine deiminases family, the exposed guanidine groups existing in a polypeptide chain are deiminated. As a consequence, ammonia is released and the citrulline residue replaces arginine in the protein structure. The deimination comprise the integral part of the mechanisms, which occur in the human body during keratinization and hair formation processes.When non-physiological substrates of are subjected to citrullination by peptidyl arginine deiminases, products of the reaction include citrulline residues is the sequence that can be recognized as foreign antigens. This can lead to the development of autoimminological diseases, e.g. rheumatoid arthritis. It was proved before, that the persons with diagnosed rheumatoid arthritis have got elevated level of peptidyl arginine deiminase type 2 and type 4 in the disease changed joints. There is a significant interrelationship between periodontitis and rheumatoid arthritis can be linked by the activity of the Porphyromonas gingivalis peptidyl deiminase. The deimination of antibacterial peptides can alter their function because of change of overall charge of a molecule. The results obtained in this thesis prove, that indeed the cathelicidine LL-37, as well as mouse peptide CRAMP and short form of LL 37(17 37) are losing the antimicrobial properties as a result of incubation with the rabbit muscle deiminase and recombinant human peptidyl arginine deiminase 2. In addition, LL-37 ability to bind LPS endotoxin is lost as a result of arginine residues deimination