LÄHDE:
Front Cell Infect Microbiol. 2017 Mar 6;7:54. doi: 10.3389/fcimb.2017.00054. eCollection 2017.
Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors.
Price C1, Merchant M2, Jones S1, Best A1, Von Dwingelo J1, Lawrenz MB3, Alam N4, Schueler-Furman O4, Kwaik YA3.
Abstraktin suomennos.
FIH-välitteinen posttranslationaalinen modifikaatio Asn--hydroxylaatiolla merkitsee eukaryoottisille proteiiniella proteiini-proteiini-interaktiossa olennaista seikkaa.
Yhdestätoista Legionella pneumophilasta (siis sen translokoituneista effektoreista) on identifioitu FIH-tunnistusmotiivi , yersiniasta YopM, Shigellasta IpaH4.5 ja Ricketssiasta ankyriiniproteiini.
Massapsketromeriset analyysit L. opneumophilan AnkB ja AnkH effektoriproteiinista vahvastiaa , että niisä on tapahtunut asparainyylihydroksylaatio.
AnkB.effektoria lokalisoituu Lpn -bakteeria sisältävään LCV vakuoliin ja se osoittautuu FIH-modifiaation saaneeksi. Isäntäsolun proteiineista sen kanssa interaktion tekevät Mint2 ja MT1-MMP, joita legionellaa sisältävä vakuoli (LCV) vaatii Dot/Icm tyyppi IV-sekreetiosta riippuvalla tavalla.
Jos estetään FHI kemiallisesti tai RNAi-välitteisellä FHI poistogeenisyydellä , legionella pneumophilan intravakuolaarinen replikaatio kumoutuu. Nämä tiedot osoittavat, että patogeenia sisältävä vakuoli hankkii isäntäkehosta FIH ja että asparaginyylihydroksylaatio on välttämätön translokoituneille efektoriproteiineille , jota ne voisivat olla funktionaalisia.
- Abstract
- FIH-mediated
post-translational modification through asparaginyl hydroxylation of
eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing
vacuole (LCV) membrane and its modification by FIH, our data show that
FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by
the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function.KEYWORDS: AnkB; Dot/Icm; FIH; Legionella; ankyrin; asparagine hydroxylation; bacterial pathogenesis; hypoxia-inducible factor (HIF).
Mikä on FIH-välitteinen posttranslationaalinen modifikaatio asn-hydroksylaatiolla?
https://www.frontiersin.org/articles/10.3389/fcimb.2017.00054/full
---Citate:
Although intracellular bacterial pathogens have been shown to exploit various host post-translational machineries, their exploitation of the host asparaginyl hydroxylation post-translational modification has never been described. The 2-oxoglutarate dioxygenase, designated as factor inhibiting HIF1 (FIH), is a key eukaryotic enzyme, which selectively hydroxylates an asparagine residue within the L(X)5[D/E]φNφ motif (φ represents aliphatic amino acids) in eukaryotic proteins (Hewitson et al., 2002; Lando et al., 2002a,b; Cockman et al., 2009). The addition of the strongly electronegative oxygen atom increases both polarity of a protein and can act as a hydrogen bond donor and acceptor. Therefore, hydroxylation can function as a “molecular switch” for protein-protein interactions (Loenarz and Schofield, 2011). FIH plays a key role in various cellular processes and in particular, it regulates the activity of hypoxia-inducible factor (HIF1), which is the master transcriptional regulator of hypoxia (Webb et al., 2009). During normoxia, HIF1 is hydroxylated by FIH on an asparagine residue and this modification acts as a molecular switch to prevent interaction with its co-activator p300/CBP, blocking transcription of hundreds of HIF1-regulated genes involved in oxygen homeostasis, energy production and immune responses (Hewitson et al., 2002; Lando et al., 2002a,b). In addition, FIH catalyzes asparaginyl hydroxylation of approximately 20 ankyrin repeat domain-containing (ARD) proteins such as p105 and IκBα (Cockman et al., 2009). FIH-dependent hydroxylation of the ARD protein, ASPP2, is required for binding of this protein to its target Par-3 (Janke et al., 2013). Therefore, asparaginyl hydroxylation acts as a molecular switch to promote or reduce protein-protein interactions between HIF1-p300/CBP and ASPP2-Par3 (Hewitson et al., 2002; Lando et al., 2002a,b; Janke et al., 2013). Additionally, FIH hydroxylates the deubiquitinase OTUB, which appears to regulate cellular metabolism (Scholz et al., 2016). A recent study has revealed a complex FIH interactome with many proteins that may serve as substrates for FIH enzyme activity, thus greatly expanding the number of eukaryotic proteins modified by asparaginyl hydroxylation (Rodriguez et al., 2016). However, the biological consequence of asparaginyl hydroxylation of eukaryotic proteins largely remains unclear.
When L. pneumophila invades amoebae or human macrophages, it evades the default endosomal-lysosomal degradation pathway and remodels its phagosome into a specialized ER-derived vacuole via intercepting ER-to-golgi vesicular traffic (Isberg et al., 2009; Al-Quadan et al., 2012; Price et al., 2014). This is achieved by the translocation of ~300 effector proteins via the Dot/Icm type IVB secretion system T4SS (de Felipe et al., 2008; Isberg et al., 2009; Zhu et al., 2011). These effectors modulate a myriad of eukaryotic processes including host signaling, vesicular trafficking, protein synthesis, apoptosis, prenylation, ubiquitination, and proteasomal degradation (Al-Quadan et al., 2012; Price et al., 2014). Surprisingly, very few of these effectors are essential for intracellular replication of L. pneumophila, suggesting specific requirements for different effectors in different environmental hosts.
The AnkB translocated effector is essential for proliferation of L. pneumophila within the two evolutionarily-distant hosts, mammalian and protozoan cells, and for intrapulmonary bacterial proliferation and manifestation of pulmonary disease in the mouse model (Al-Khodor et al., 2008; Price et al., 2009, 2010a,b, 2011; Lomma et al., 2010). Recent characterization of the crystal structure of AnkB has confirmed that it is a non-canonical F-box protein with three ankyrins repeats domain (Price et al., 2009; Lomma et al., 2010; Wong et al., 2017). The crystal structure has also confirmed that the F-box domain of AnkB interacts with the host SCF1 ubiquitin ligase, which explains show AnkB functions as a platform for the docking of polyubiquitinated proteins to the Legionella-containing vacuolar (LCV) membrane within macrophages and amoebae (Price et al., 2009; Lomma et al., 2010; Wong et al., 2017). The AnkB-assembled polyubiquitinated proteins are predominately Lys48-linked that are ultimately degraded by the host proteasome machinery, which generates higher levels of cellular amino acids that are the main sources of carbon and energy to power replication of L. pneumophila (Price et al., 2011). This enables intracellular bacteria to overcome host limitation of essential nutrients and favorable sources of carbon and energy, such as amino acids (Price et al., 2011; Abu Kwaik and Bumann, 2013).
- ( Tässä johdetaan solussa hajoitettavaksi tuomitut eli K48 polyubikitinoidut jäteproteiinit LCV vakuoliin, jossa Legionella käyttää niiden aineksen replikaatioon tarvittavana ravintona- asiaa ei solu varmaan pysty "huomaamaan").
Here we show that 11 L. pneumophila type IVB-translocated effectors including, AnkB and AnkH, harbor the recognition motif for FIH-dependent asparaginyl-hydroxylation. Furthermore, the FIH recognition motif is found in translocated effectors from other intracellular microbial pathogens including YopM from Yersinia pestis, IpaH4.5 of Shigella flexneri and a putative translocated ARD-protein of Rickettsia felis. We show that the AnkH and AnkB effectors are modified by asparaginyl hydroxylation. The LCV recruits FIH, which is indispensable for intra-vacuolar proliferation of L. pneumophila and plays a partial role in the ability of the LCV to evade lysosomal fusion and is needed for AnkB-dependent assembly of polyubiquitinated proteins on the LCV. This is the first example of an injected microbial effectors post-translationally modified by asparaginyl hydroxylation.
Acquisition of FIH, Mint3, and MT1-MMP by the LCV
The AnkB effector is localized to the LCV membrane through host-mediated farnesylation (Price et al., 2010b). In addition, the effectors LepB, SdeC, and SdcA which are potential candidates for FIH-mediated asparaginyl hydroxylation are also LCV-localized (Chen et al., 2004, 2007; Luo and Isberg, 2004; Bardill et al., 2005; Ingmundson et al., 2007; Tan et al., 2011). Since FIH is a cytosolic enzyme that can be sequestered to membranous structures such as the Golgi apparatus through interaction with Mint3 and MT1-MMP in macrophages (Sakamoto and Seiki, 2009, 2010) and the LCV intercepts ER-Golgi vesicular traffic (Isberg et al., 2009; Al-Quadan et al., 2012; Price et al., 2014), we determined if FIH and its two interacting partners (Mint3 and MT1-MMP) were recruited to the LCV within hMDMs.The data showed that by 2 h of infection, 75, 65, and 56% of the LCVs harboring wild type bacteria co-localized with FIH, Mint3, and MT1-MMP, respectively (Figures 2A–D). In contrast, only 27, 18.2, and 0% of the LCVs harboring the dotA translocation-deficient mutant co-localized with FIH, Mint3, and MT1-MMP, respectively (Figures 2A–D) and this was significantly reduced relative to co-localization observed for wild type LCVs (unpaired t-test, p < 0.01). This indicates recruitment of these proteins to the LCV is dependent on the Dot/Icm T4SS apparatus.- Jotta tämä F1H funktio saataisiin pyydystettyä LCV vakuoleihin, legionella T5SS systeemin on rekrytoitava myös Mint3 ja MT1-MMP, koska F1H on sytosolinen entsyymi ja näiden toisten tekijöiden avulla se saadaan saostumaan kalvoihin kuten ER-Golgi, jolloin se on legionellan tarpeisiin saatavissa ja sijoitettavissa LCV vakuoleihin.
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AnkB plays a central role for L. pneumophila by promoting the degradation of polyubiquitinated proteins which allows this organism to access essential amino acids that are used for both energy and a carbon source (Price et al., 2009, 2011). Substitution of the three hydroxylated asparagine residues significantly impacts the ability of AnkB to recruit polyubiquitinated proteins to the LCV and concomitantly fails to restore intra-vacuolar replication of an ankB mutant strain of L. pneumophila.
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Furthermore, blocking host FIH activity results in a similar phenotype to the AnkB substitutions, and taken together suggests that asparaginyl hydroxylation of AnkB contributes to the function of this effector.
Blocking FIH activity results in a dose-dependent inhibition of intra-vacuolar replication of L. pneumophila. Interestingly however, only ~55% of LCVs trafficked to a lysosomal compartment, indicating that the FIH-mediated block in intra-vacuolar replication of L. pneumophila has both lysosomal evasion-independent and -dependent mechanisms.
Both AnkB and AnkH are needed for intra-vacuolar proliferation of L. pneumophila but neither impacts the normal trafficking and biogenesis of the LCV (Al-Khodor et al., 2008; Habyarimana et al., 2008, 2010; Price et al., 2009, 2010a,b, 2011; Lomma et al., 2010).
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The lysosomal evasion-dependent mechanism may involve both injected effectors, though no single injected effector to date has been shown to be required for the ability of the LCV to evade the lysosomes (de Felipe et al., 2008; Isberg et al., 2009; Zhu et al., 2011).
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Mint3
In macrophages, membrane-associated FIH is inactive, at least in terms of HIF1 hydroxylation activity, but through its binding to Mint3 it enables HIF1 to promote transcription of glycolytic genes that are needed by the macrophage to generate ATP (Sakamoto and Seiki, 2009, 2010).
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L. pneumophila uses host amino acids as the primary source of carbon and energy by AnkB-dependent proteasomal degradation, but exogenous pyruvate alone can compensate for proteasomal degradation to enable intra-vacuolar replication of L. pneumophila (Price et al., 2011). This indicates that host pyruvate is an additional metabolite scavenged by intra-vacuolar L. pneumophila. Therefore, a consequence of FIH recruitment to the LCV may be increased HIF1 activity, which will ultimately increase availability of pyruvate that the bacteria can scavenge from the intracellular environment to use as an energy source and building block of macromolecules.
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