Etiketter

onsdag 10 oktober 2018

"MMP, Autophagosome" ( PubMed Haku)

https://www.ncbi.nlm.nih.gov/pubmed/?term=MMP%2C+Autophagosome

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Items: 1 to 20 of 44


 Latkripiini 1
 
2.
LP1 from Lentinula edodes C91-3 Induces Autophagy, Apoptosis and Reduces Metastasis in Human Gastric Cancer Cell Line SGC-7901.
Batool S, Joseph TP, Hussain M, Vuai MS, Khinsar KH, Din SRU, Padhiar AA, Zhong M, Ning A, Zhang W, Cao J, Huang M.
Int J Mol Sci. 2018 Sep 30;19(10). pii: E2986. doi: 10.3390/ijms19102986.PMID:30274346
Free Article
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Present study aimed to elucidate the anticancer effect and the possible molecular mechanism underlying the action of Latcripin 1 (LP1), from the mushroom Lentinula edodes strain C91-3 against gastric cancer cell lines SGC-7901 and BGC-823. Cell viability was measured by Cell Counting Kit-8 (CCK-8); morphological changes were observed by phase contrast microscope; autophagy was determined by transmission electron microscope and fluorescence microscope. Apoptosis and cell cycle were assessed by flow cytometer; wound-healing, transwell migration and invasion assays were performed to investigate the effect of LP1 on gastric cancer cell's migration and invasion. Herein, we found that LP1 resulted in the induction of autophagy by the formation of autophagosomes and conversion of light chain 3 (LC3I into LC3II.
 LP1 up-regulated the expression level of autophagy-related gene (Atg7, Atg5, Atg12, Atg14) and Beclin1; increased and decreased the expression level of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins respectively, along with the activation of Caspase-3.
 At lower-doses, LP1 have shown to arrest cells in the S phase of the cell cycle and decreased the expression level of matrix metalloproteinase MMP-2 and MMP-9.
 In addition, it has also been shown to regulate the phosphorylation of one of the most hampered gastric cancer pathway, that is, protein kinase B/mammalian target of rapamycin (Akt/mTOR) channel and resulted in cell death.
These findings suggested LP1 as a potential natural anti-cancer agent, for exploring the gastric cancer therapies and as a contender for further in vitro and in vivo investigations.KEYWORDS:
Latcripin 1; Lentinula edodes; apoptosis; autophagy; gastric cancer; metastasis

 
3.
MgO nanoparticles cytotoxicity caused primarily by GSH depletion in human lung epithelial cells.
Akhtar MJ, Ahamed M, Alhadlaq HA, Alrokayan SA.
J Trace Elem Med Biol. 2018 Dec;50:283-290. doi: 10.1016/j.jtemb.2018.07.016. Epub 2018 Jul 20.PMID:30262293

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4.
Inhibiting autophagy promotes collagen degradation by regulating matrix metalloproteinases in pancreatic stellate cells.
Li CX, Cui LH, Zhuo YZ, Hu JG, Cui NQ, Zhang SK.
Life Sci. 2018 Sep 1;208:276-283. doi: 10.1016/j.lfs.2018.07.049. Epub 2018 Jul 26.PMID:30056017
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AbstractAIMS:Autophagy is an intracellular metabolic process that degrades and recycles own constituents to maintain homeostasis and supply substrates. Disruption of collagen degradation is one of the pathogenesis of pancreatic fibrosis. In this study, we investigated the effects of inhibiting autophagy on the collagen degradation of PSCs. MAIN METHODS:
...  Autophagosome was confirmed by transmission electron microscope. Immunofluorescence for LC3B and α-SMA were applied to assess autophagy and activated PSCs. The effects of autophagy inhibition of 3-MA on the expressions of LC3B, Atg5, and Beclin-1 were investigated by real-time PCR and Western blotting, as well as the α-SMA, TGF-β1, ColI, Col III, FN, MMP-2, MMP-13, TIMP-1 and TIMP-2. Meanwhile, the secretion of ColI, Col III and FN were investigated by ELISA. KEY FINDINGS:
The LC3-II/I ratio was increased in rat CP model. Autophagosomes and an increased autophagic level were observed during PSCs activation. Inhibiting autophagy could down-regulate the expressions of α-SMA, TGF-β1, FN, ColI, Col III, TIMP-1 and TIMP-2, while the expressions of MMP-2 and MMP-13 were increased. SIGNIFICANCE: This study confirmed that autophagic level is increased during PSCs activation in vivo and in vitro. Inhibiting autophagy prevents the activation of PSCs, and suppresses fibrosis through promoting extracellular matrix (ECM) degradation by decreasing the expression of TGF-β1 and increasing MMPs/TIMPs ratio.Copyright © 2018 Elsevier Inc. All rights reserved.KEYWORDS:
Autophagy; Collagen; LC3B; Pancreatic stellate cells; α-SMA

 
5.
Diazoxide ameliorates severity of experimental osteoarthritis by activating autophagy via modulation of the osteoarthritis-related biomarkers.
Meng Z, Shen B, Gu Y, Wu Z, Yao J, Bian Y, Zeng D, Chen K, Cheng S, Fu J, Peng L, Zhao Y.
J Cell Biochem. 2018 Jun 28. doi: 10.1002/jcb.27145. [Epub ahead of print] PMID: 29953665
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Accumulating evidence suggests that autophagy plays a protective role in chondrocytes and prevents cartilage degeneration in osteoarthritis (OA). The objective of this study was to investigate the effect of diazoxide on chondrocyte death and cartilage degeneration and to determine whether these effects are correlated to autophagy in experimental OA. In this study, a cellular OA model was established by stimulating SW1353 cells with interleukin 1β. A rat OA model was generated by transecting the anterior cruciate ligament combined with the resection of the medial menisci, followed by treatment with diazoxide or diazoxide combination with 3-methyladenine. The percentage of viable cells was evaluated using calcein-acetoxymethyl/propidium iodide double staining. The messenger RNA expression levels of collagen type II alpha 1 chain (COL2A1), matrix metalloproteinase 13 (MMP-13), TIMP metallopeptidase inhibitor 1 (TIMP-1), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were determined using quantitative real-time polymerase chain reaction. The cartilage thickness and joint space were evaluated using ultrasound. SW1353 cell degeneration and autophagosomes were observed using transmission electron microscopy. The expression levels of microtubule-associated protein 1 light chain 3 (LC3), beclin-1, P62, COL2A1, and MMP-13 were evaluated using immunofluorescence staining and Western blot analysis. Diazoxide significantly attenuated articular cartilage degeneration and SW1353 cell death in experimental OA. The restoration of autophagy was observed in the diazoxide-treated group. The beneficial effects of diazoxide were markedly blocked by 3-methyladenine. Diazoxide treatment also modulated the expression levels of OA-related biomarkers. These results demonstrated that diazoxide exerted a chondroprotective effect and attenuated cartilage degeneration by restoring autophagy via modulation of OA-related biomarkers in experimental OA. Diazoxide treatment might be a promising therapeutic approach to prevent the development of OA.KEYWORDS:
SW1353 cell; autophagy; diazoxide; osteoarthritis; osteoarthritis-related biomarker

(Ajatuksia herättävä artikkeli: siis  modulointia  solukaliumin ja kalsiumin suhteen  nivelrustotasossa!)
 

7.
BST106 Protects against Cartilage Damage by Inhibition of Apoptosis and Enhancement of Autophagy in Osteoarthritic Rats.
Hong JM, Shin JK, Kim JY, Jang MJ, Park SK, Lee JH, Choi JH, Lee SM.
Biol Pharm Bull. 2018 Aug 1;41(8):1257-1268. doi: 10.1248/bpb.b18-00207. Epub 2018 May 23.
PMID:
29794403
Free Article
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8.
Physakengose G induces apoptosis via EGFR/mTOR signaling and inhibits autophagic flux in human osteosarcoma cells.
Lin H, Zhang C, Zhang H, Xia YZ, Zhang CY, Luo J, Yang L, Kong LY.
Phytomedicine. 2018 Mar 15;42:190-198. doi: 10.1016/j.phymed.2018.03.046. Epub 2018 Mar 19.
PMID:
29655686
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9.
Autophagy attenuates copper-induced mitochondrial dysfunction by regulating oxidative stress in chicken hepatocytes.
Yang F, Liao J, Pei R, Yu W, Han Q, Li Y, Guo J, Hu L, Pan J, Tang Z.
Chemosphere. 2018 Aug;204:36-43. doi: 10.1016/j.chemosphere.2018.03.192. Epub 2018 Mar 29.
PMID:
29649662
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10.
Brevilin A induces apoptosis and autophagy of colon adenocarcinoma cell CT26 via mitochondrial pathway and PI3K/AKT/mTOR inactivation.
You P, Wu H, Deng M, Peng J, Li F, Yang Y.
Biomed Pharmacother. 2018 Feb;98:619-625. doi: 10.1016/j.biopha.2017.12.057. Epub 2017 Dec 29.
PMID:
29289836
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11.
Unsaturated fatty acids protect trophoblast cells from saturated fatty acid-induced autophagy defects.
Hong YJ, Ahn HJ, Shin J, Lee JH, Kim JH, Park HW, Lee SK.
J Reprod Immunol. 2018 Feb;125:56-63. doi: 10.1016/j.jri.2017.12.001. Epub 2017 Dec 14.
PMID:
29253794
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12.
Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses.
Lombardo T, Folgar MG, Salaverry L, Rey-Roldán E, Alvarez EM, Carreras MC, Kornblihtt L, Blanco GA.
Basic Clin Pharmacol Toxicol. 2018 May;122(5):489-500. doi: 10.1111/bcpt.12945. Epub 2018 Jan 3.
PMID:
29205851
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13.
ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells.
Lv W, Sui L, Yan X, Xie H, Jiang L, Geng C, Li Q, Yao X, Kong Y, Cao J.
Chem Biol Interact. 2018 Jan 5;279:136-144. doi: 10.1016/j.cbi.2017.11.013. Epub 2017 Nov 24.
PMID:
29179951
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14.
Mdivi-1 alleviates blood-brain barrier disruption and cell death in experimental traumatic brain injury by mitigating autophagy dysfunction and mitophagy activation.
Wu Q, Gao C, Wang H, Zhang X, Li Q, Gu Z, Shi X, Cui Y, Wang T, Chen X, Wang X, Luo C, Tao L.
Int J Biochem Cell Biol. 2018 Jan;94:44-55. doi: 10.1016/j.biocel.2017.11.007. Epub 2017 Nov 22.
PMID:
29174311
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15.
Trolline Ameliorates Liver Fibrosis by Inhibiting the NF-κB Pathway, Promoting HSC Apoptosis and Suppressing Autophagy.
Bai F, Huang Q, Nie J, Lu S, Lu C, Zhu X, Wang Y, Zhuo L, Lu Z, Lin X.
Cell Physiol Biochem. 2017;44(2):436-446. doi: 10.1159/000485009. Epub 2017 Nov 15.
PMID:
29141243
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16.
A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1β-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.
Ansari MY, Khan NM, Haqqi TM.
Biomed Pharmacother. 2017 Dec;96:198-207. doi: 10.1016/j.biopha.2017.09.140. Epub 2017 Oct 6.
PMID:
28987943
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17.
Exogenous H2S Protects Against Diabetic Cardiomyopathy by Activating Autophagy via the AMPK/mTOR Pathway.
Yang F, Zhang L, Gao Z, Sun X, Yu M, Dong S, Wu J, Zhao Y, Xu C, Zhang W, Lu F.
Cell Physiol Biochem. 2017;43(3):1168-1187. doi: 10.1159/000481758. Epub 2017 Oct 5.
PMID:
28977784
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18.
Protective effect of madecassoside on H2O2-induced oxidative stress and autophagy activation in human melanocytes.
Ling Y, Gong Q, Xiong X, Sun L, Zhao W, Zhu W, Lu Y.
Oncotarget. 2017 May 7;8(31):51066-51075. doi: 10.18632/oncotarget.17654. eCollection 2017 Aug 1.
PMID:
28881630
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19.
Protoporphyrin IX-mediated sonodynamic therapy promotes autophagy in vascular smooth muscle cells.
Li Q, Kang J, Xiong X, Liu Y, Cao W, Liu Y, Li Y.
Oncol Lett. 2017 Aug;14(2):2097-2102. doi: 10.3892/ol.2017.6394. Epub 2017 Jun 16.
PMID:
28789437
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20.
FOXO3 promoted mitophagy via nuclear retention induced by manganese chloride in SH-SY5Y cells.
Song D, Ma J, Chen L, Guo C, Zhang Y, Chen T, Zhang S, Zhu Z, Tian L, Niu P.
Metallomics. 2017 Sep 20;9(9):1251-1259. doi: 10.1039/c7mt00085e.
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28661534
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Autofagosomibiogeneesi (2013 )( mitokondria, syntaxiini17 etc, starvaatio )

https://www.nature.com/articles/cr2013159#f2

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Miten Legionella LCV pääse irti mitokondriasta, johon se ajautuu , ja nitistää Syntaxiinin17 ja miksi.

LCV vakuolin  tutkimuksissa on havaittu sen joutuvan mitokondriokontaktiin  hetkiseksi ja sitten irtoavan välttäen endosyyttisiä  kuljetusteitä ja liittyen  ER-Golgi retrogradiseen  kalvomateriaaliin.
 Tästä löytyy selvitys viime vuoden artikkelissa, toukokuu 2017.

Legionellan effektoriproteiini Lpg1137 katkaisee  ER- mitokondriakommunikaation pilkkomalla syntaksiini17- proteiinin. 

LÄHDE: 
 https://www.ncbi.nlm.nih.gov/pubmed/28504273
Nat Commun. 2017 May 15;8:15406. doi: 10.1038/ncomms15406.
Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17. Arasaki K1, Mikami Y1, Shames SR2, Inoue H1, Wakana Y1, Tagaya M1

Tiivistelmästä suomennosta.

Makrofagin infektoitumisen aikana patogeeninen legionella pneumophilabakteeri erittää effektoriproteiineja, jotka indusoivat  konversion  plasmakalvoperäisesta  vakuolista  endoplasmisen retikulumin kaltaiseksi replikatiiviseksi vakuoliksi. Nämä  endoplasmista  verkostoa (ER)  muistuttavat vakuolit (LCV)  lopuksi fuusioituvat endoplasmiseen verkostoon (ER), jossa patogeeni replikoituu.
Tässä työssään tutkijat osoittavat, että L. pneumophilan efsektori Lpg1137  on eräs seriiniproteaasi, jonka vaikutuskohde  on  mitokondria ja siihen assosioituneet kalvot.
 Lpg1137 sitoutuu  syntaxiin 17 ja pilkkoo sen.
  Syntaxiini 17 on eräs liukoinen N-etyylimaleimidi-sensitiiviseen faktoriin liittyvä proteiinireseptori (SNARE) -proteiini, jonka tiedetään osallistuvan  mitokondrian dynamiikan säätelyyn tekemällä interaktio mitokondrian fissiofaktorin Drp1 kanssa   ravintoa saavissa soluissa ja  autofagiaan  Atg14L:n  ja muiden SNARE-proteiinien  kanssa  nälkätilassa olevassa solussa.
 Kun  Syntaxiini 17 pilkkoutuu,  estyy  sekä autofagia että stauroporiinilla indusoituva  apoptoosi, jota Bax, Drp-1:stä riippuvalla tavalla tapahtuu.
Täten Legionella pneumophila voi tukkia  ER- mitokondria - kommunikaation pilkkomalla syntaxiini17 -proteiinin.

  • During infection of macrophages, the pathogenic bacterium Legionella pneumophila secretes effector proteins that induce the conversion of the plasma membrane-derived vacuole into an endoplasmic reticulum (ER)-like replicative vacuole. These ER-like vacuoles are ultimately fused with the ER, where the pathogen replicates. Here we show that the L. pneumophila effector Lpg1137 is a serine protease that targets the mitochondria and their associated membranes. Lpg1137 binds to and cleaves syntaxin 17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that is known to participate in the regulation of mitochondrial dynamics through interaction with the mitochondrial fission factor Drp1 in fed cells and in autophagy through interaction with Atg14L and other SNAREs in starved cells. Cleavage of syntaxin 17 inhibits not only autophagy but also staurosporine-induced apoptosis occurring in a Bax, Drp1-dependent manner. Thus, L. pneumophila can shut down ER-mitochondria communication through cleavage of syntaxin 17. PMID:28504273 PMCID: PMC5440676 DOI: 10.1038/ncomms15406
Free PMC Article
Sitaatti yksityiskohdasta.

Syntaxin 17 (Stx17) is a SNARE originally implicated in a vesicle-trafficking step to the smooth-surfaced tubular ER membranes that are abundant in steroidogenic cells14.

 Stx17 is unique in that it has a long hairpin-like C-terminal hydrophobic domain (CHD), followed by a cytoplasmic basic region. Stx17 participates in cellular events unrelated to membrane fusion.

 In fed cells, Stx17 promotes mitochondrial fission by defining the localization and activity of the mitochondrial fission factor Drp1 (ref. 15).

 On starvation, on the other hand, Stx17 dissociates from Drp1 and associates with Atg14L, a subunit of the phosphatidylinositol 3-kinase (PI3K)  complex. This promotes the recruitment of this kinase to the mitochondria-associated ER membrane (MAM)16, which leads to the formation of phosphatidylinositol 3-phophate (PI3P)-enriched omegasomes that are considered to represent a membrane/lipid source for autophagosomes17,18. 
In the late stage of autophagy, Stx17 present on autophagosomes mediates the fusion of autophagosomes with lysosomes19,20,21.
In this study, we show that Stx17 is degraded on Legionella infection. We identify the Legionella effector Lpg1137 as the responsible protein for Stx17 breakdown and show that Lpg1137 is a serine protease that localizes to the ER–mitochondria contact site, where Stx17 is located.
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Ihmisen MMP entsyymit voivat aktivoitua bakteerien proteinaaseilla

http://www.jbc.org/content/272/9/6059.full.html
Activation of Human Matrix Metalloproteinases by Various Bacterial Proteinases*

Tatsuya Okamoto‡,§, Takaaki Akaike‡, Moritaka Suga§,
Sumio Tanase, Hidechika Horie‡, Seiya Miyajima‡,
Masayuki Ando§, Yoshio Ichinose∥ and Hiroshi Maeda‡**
  1. From the Department of ‡ Microbiology
  2. § Internal Medicine I, and
  3. Biochemistry II, Kumamoto University School of Medicine, Kumamoto 860 and the
  4. ∥ Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan
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Legionellan effektoriproteiini AnkB vaatii isäntäsolulta FIH/ asparaginyylihydroksylaation

https://www.karger.com/Article/Pdf/235770


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.

    ---- Some citates from the article : 
    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.
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    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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    Original Research ARTICLE

    Front. Cell. Infect. Microbiol., 06 March 2017 | https://doi.org/10.3389/fcimb.2017.00054

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