細(xì)胞生物學(xué)文獻(xiàn)項(xiàng)目匯報(bào)

1、細(xì)胞生物學(xué)文獻(xiàn)項(xiàng)目匯報(bào)Human IRGM Induces Autophagy toEliminate Intracellular Mycobacteria Sudha B. Singh, Alexander S. Davis, Gregory A. Taylor,Vojo Deretic Science 313, 1438 (2006)Introduction 1、AutophagyAutophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. D

2、espite its simplicity, recent progress has demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles, which are sometimes complex.Autophagy consists of several sequential stepssequestration,transport to lysosomes, degradation, and Utilization of degradation produ

3、ctsand each step may exert different function. These step-dependent functions may allow autophagy to be multifunctional. Autophagy is a general term for the degradation of cytoplasmic components within lysosomes. This process is quite distinct from endocytosis-mediated lysosomal degradation of extra

4、cellular and plasma membrane proteins. There are three types of autophagymacroautophagy, microautophagy, and chaperone-mediated autophagy and the term “autophagy” usually indicates macroautophagy unless otherwise specified (Fig.1). Autophagy is mediated by a unique organelle called the autophagosome

5、. As autophagosomes engulf a portion of cytoplasm, autophagy is generally thought to be a nonselective degradation system. This feature is in marked contrast to the ubiquitinproteasome system, which specifically recognizes only ubiquitinated proteins for proteasomal degradation. It is therefore reas

6、onable to assume that the ubiquitinproteasome system has numerous specific functions because it can selectively degrade thousands of substrates.Figure 1. The process of macroautophagy in mammalian cells. A portion of cytoplasm, including organelles, is enclosed by a phagophore or isolation membrane

7、to form an autophagosome. The outer membrane of the autophagosome subsequently fuses with the endosome and then the lysosome, and the internal material is degraded. In yeast, autophagosomes are generated from the PAS, which has not yet been identified in mammalian cells. The nomenclature for various

8、 autophagic structures is indicated.However, recent studies have clearly demonstrated that autophagy has a greater variety of physiological and pathophysiological roles than expected, such as starvation adaptation, intracellular protein and organelle clearance, development, anti-aging, elimination o

9、f microorganisms, cell death, tumor suppression, and antigen presentation. Additionally, in some situations, the contribution of autophagy seems to be very complicated.For example, it is very difficult to generalize the role of autophagy in cancer and cell death.This is like the question: “Is inflam

10、mation good or bad for life?” Of course, inflammation is required for the anti-bacterial response, but the inflammatory response associated with bacterial pneumonia can be life-threatening.Therefore, it may be difficult to draw simplified connections between autophagy and higher-order functions.To u

11、nderstand the various roles of autophagy, it may be useful to subclassify macroautophagy into “induced autophagy” and “basal autophagy”.The former is used to produce amino acids following starvation, while the latter is important for constitutive turnover of cytosolic components. However, even this

12、distinction is too simplified and cannot be applied to more complicated issues. IRGMThe immunity-related guanosine triphosphatases (GTPases) 鳥嘌呤三磷酸酶or IRGs, also known as p47 GTPases , play a role in innate immunity against intracellular pathogens. There are 23 complete Irg genes in the mouse genome

13、, whereas only 3 identifiable IRG genes are seen in the human genome. Murine IRG GTPases play a role in the control of intracellular pathogens,but the functional role, if any, of human IRGs is unclear. Furthermore, most murine Irg genes are inducible by interferon-g (IFN-), whereas the human IRG gen

14、es are not . In mice,IIGP1 (Irga6) affects the integrity of vacuoles in Toxoplasma, whereas LRG-47 (Irgm1), acting in defense against intracellular Mycobacterium tuberculosis , may participate in IFN-induced autophagy 。Autophagy is a cellular homeostasis mechanism, whereby portions of the cytosol an

15、d damaged organelles or intracellular pathogens and their products are sequestered into an autophagosome for degradation in autolysosomes. We investigated the role of the murine Irgm1 GTPase and its putative human ortholog IRGM in autophagy and tested whether human IRGM plays a role in control of in

16、tracellular mycobacteria.Materials and MethodsCell and Bacterial culturesThe murine macrophage cell line RAW 264.7 was maintained in DME supplemented with 4 mM L-glutamine and 10% fetal bovine serum (FBS). Human U937 cells were grown in RPMI 1640 supplemented with 2mM L-glutamine, 4.5 g/L glucose, 1

17、0% FBS, 10mM HEPES, and 1mM sodium pyruvate. U937 cells were allowed to differentiate by adding phorbol 12-myristate 13-acetate. 293T cells and HeLa cells were maintained in DME supplemented with 4mM Lglutamine, 100 U/ml penicillin, 100 g/ml of streptomycin, and 10% FBS. Mycobacterium tuberculosis v

18、ar. bovis BCG was grown in Middlebrook 7H9 broth, and BCG expressing GFP was maintained in 7H9 broth containing 25 g/ml kanamycin.Preparation of Texas-Red labeled Mycobacterium tuberculosis var. bovis BCGMycobacterium tuberculosis var. bovis BCG was grown in Middlebrook 7H9 broth and labeled in 0.5

19、mg/ml Texas Red-succinimidyl ester (Molecular Probes) in neutral phosphate-buffered saline (PBS) for 1 h. Labeled mycobacteria were washed three times in PBS, homogenized to generate single cell suspensions and then opsonized in DME supplemented with 10% FBS.Plasmid constructs and transfectionpEGFP-

20、LC3 was from Drs. Noboru Mizushima and Tamotsu Yoshimori (National Institute for Basic Biology, Okazaki, Japan). The plasmid construct GFP-LRG47 (GFP-Irgm1) was from Dr. Greg Taylor (Duke University Medical Center Durham,North Carolina). Transfection of U937 cells or RAW 264.7 cells was carried out

21、as previously described. 293T cells were transfected using a calcium phosphate kit (Invitrogen) and HeLa cells were transfected using the Effectene Transfection Reagent (Qiagen) both according to manufacturers protocol.Antibodies, cytokines, and fluorescent dyesRabbit polyclonal antibody to CD63 was

22、 from Santa Cruz Biotechnology. Rabbit polyclonal antibody against LC3 was a gift from Takashi Ueno (Juntendo University School of Medicine, Japan). Monoclonal antibody against GAPDH was from Abcam. Anti-IRGM polyclonal antibody against a synthetic peptide corresponding to the C-terminal amino acids

23、 of IRGM and an additional cysteine (CQIRENVLENLQKER) was prepared by immunization of rabbits and affinity purified. Murine IFN-, 3-Methyladenine, Rapamycin and Wortmannin were purchased from Sigma. Staining with the acidotropic dye LysoTracker Red DND- 99 (Molecular Probes, Eugene, OR) was carried

24、out as previously described。 Monodansylcadaverine (MDC) was procured from Fluka and used as previously described. DQ Red BSA and secondary antibodies conjugated to Alexa 647 were from Molecular Probes. DQ Red BSA was preloaded in the cells at 10 g/ml for 3 h. Cells were subsequently fixed and viewed

25、 using fluorescence microscopy.Induction of autophagyAutophagy was induced by treatment with rapamycin雷怕霉素(50 g/ml) for 4 h in full nutrient medium or in EBSS for 4hr. Alternatively, cells were induced for autophagy with IFN- (200 U/ml) for 24hrs.Protein knockdown by siRNAIrgm1, IRGM, Beclin1, and A

26、tg7 knockdown was achieved by using siGENOME SMARTpool reagent (Dharmacon). All effects of protein specific siRNA were compared to control Non-targeting siRNA pool (Dharmacon). Cells were transfected with 1.5 g of siRNA either by nucleoporation, calcium phosphate, or Effectene Transfection Reagent a

27、s described above for plasmid constructs.Western blottingFor immunoblotting, cells were washed in PBS and lysed with buffer containing 10 mM Tris HCl (pH 8.0), 150 mM NaCl, 0.5 % deoxycholate脫氧膽酸鹽, 2mM EDTA, 2% NP-40, 1 mM PMSF苯甲基磺酰氟化物and protease inhibitor cocktail (Roche Applied Science, Indianapo

28、lis, IN). Fifty micrograms of protein was loaded and separated on a 12 % SDSand transferred to nitrocellulose.Staining was revealed with SuperSignal West Dura chemiluminescent substrate (Pierce). GAPDH was used as a loading control.Immunofluorescence laser scanning confocal microscopy.RAW 264.7 cell

29、s grown on coverslips were fixed with 1% paraformaldehyde多聚甲醛followed by membrane permeabilization using 0.2% Saponin皂甙. After appropriate antibody incubations, coverslips were mounted using Permafluor Aqueous mounting medium (Immunon). Collection of 1 m thick optical sections was performed using an

30、 Axiovert 200M microscope with an Axioscope 63x oil objective and LSM 5 Pascal or LSM 510 META systems (Carl Zeiss). Images were cropped using Adobe Photoshop 7.0.Transmission electron microscopyTransfection of RAW 264.7 cells with GFP-Irgm1 was carried out as described above. Mock transfected cells

31、 were used as control. For IFN- treatment, cells were incubated with IFN- (200 U/ml) for 24hrs. Cells were fixed with 3% formaldehyde甲醛(from paraformaldehyde) + 2% glutaraldehyde戊二醛in 0.1 M cacodylate二甲砷酸鹽, pH 7.4. Cells were washed and postfixed in 1% osmium tetroxide, 100 mM cacodylate buffer; deh

32、ydrated with increasing concentrations of ethanol and gradually infiltrated with Epon resin環(huán)氧樹脂, embedded in straight resin and examined using a Zeiss EM 900 transmission electron microscope.Colony-Forming Unit AssayU937 cells, differentiated in 6-well plates (1106 cells per well), were infected wit

33、h 1107 mycobacteria for 1 hr at 37C, washed two times with PBS, and incubated for 30 min, 2 h, or 4 h in full medium. Cells were lysed with 1 ml of water. Quantitative culturing was performed using 10-fold serial dilutions. Aliquots of 5 l of each dilution were inoculated on Middlebrook 7H11 agar pl

34、ates with ADC. Plates were incubated for 2 weeks. For each time point, counts were made from three different wells. Colonies were counted on plates with dilutions yielding 1050 visible colonies.ResultsIFN- induces autophagy in macrophages. Irgm1 is a downstream effector of IFN- that has been implica

35、ted as a potential participant in this process. We tested the effects of Irgm1 in the absence of IFN-stimulation. Expression in macrophages of GFP-Irgm1 induced the formation of large vacuoles (3 mm) without IFN- stimulation. The Irgm1-induced profiles were positive for monodansylcadaverine丹(磺)酰戊二胺(

36、MDC), a preliminary screening marker for autophagic organelles. The proportion of cells with MDC+vacuoles in GFP-Irgm1transfected cells was 60 4% versus 12.1 1.9% in control samples (P 0.01) (Fig. 1B). The classical inhibitors of autophagy, 3-methyladenine (3MA) and wortmannin渥曼青霉素, caused a marked

37、reduction in the fraction of Irgm1 transfected cells with MDC vacuoles in cells (53.3 10.4% in samples without inhibitors versus 19 4.5% with 3MA and 21.33.5% with wortmannin; P 0.01).Fig. 1. Irgm1 induces autophagic organelles in macrophages. (A) RAW 264.7 cells were transiently transfected with GF

38、P-Irgm1 and labeled for a series of markers: 50 mM MDC for 30 min, 100 nM LT for 2 hours, and 10 mg/ml of DQ Red BSA or 500 nM MT for 30 min. Arrows indicate Irgm1-induced vacuoles that were absent in the control cells. (B) Quantification of cells with autophagic organelles that were positive for ma

39、rkers tested. Results are shown as the means (% of cells with marker positive vacuoles) T SEM; *P 0.05, *P 0.01 analysis of variance (ANOVA).(C) RAW 264.7 cells were mock transfected and treated without (I) or with (II) IFN-g. The IFN-gtreated cells with autolysosomes containing degraded material (w

40、hite arrowheads) or with double-membrane profiles in the process of engulfment of target organelles (black arrowhead) are shown. (III and IV) Cells transfected with GFP-Irgm1 show both early autophagosomes (arrows) and autolysosomes (arrowhead) that resemble the vacuoles in IFNg treated cells.Fig. S

41、2. Classical inhibitors of autophagy inhibit MDC positive structure formation induced by Irgm1. (A). RAW 264.7 cells were transiently transfected with GFP-Irgm1. Cells were treated with 3MA (10 mM) or Wortmannin (100 nM) for 2 h, followed by staining with MDC (B). Quantification of inhibition by cla

42、ssical inhibitors of autophagy. Data represents mean SEM from 3 independent experiments (*p0.05, *p0.01).The Irgm1-induced vacuolar compartments were positive for the acidotropic dye Lyso-Tracker Red (LT), indicating an acidic nature of the vacuoles. The proportion of cells transfected with Irgm1 th

43、at had LTlarge vacuoles was 62 4% versus 12 2% of control cells (P 0.01). The large Irgm1-induced vacuoles were also proteolytically active as detected by fluorophore dequenching on proteolysis of bovine serum albumin conjugated to 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (DQ Red BSA). The DQ Red

44、BSA profiles were detected in 45 5% of GFPIrgm1 transfected cells, compared to 11 1% of control cells (P 0.05) .The Irgm1-induced DQ Red BSAt vacuoles were indistinguishable from those induced by IFN-.Fig. S3. IFN- induces large vacuoles resembling those induced by Irgm1.RAW 264.7 cells were treated

45、 with IFN- for 24 h and incubated with DQ Red BSA (10 g /ml) for 4 h. Cells were washed and processed for IF using anti-Irgm1 antibody and Alexa Flour 488 conjugated secondary antibody. Arrow indicates large vacuole positive for DQ Red BSA while the arrowhead represents an empty vacuole.During forma

46、tion, autophagosomes sequester cytoplasmic contents and often include mitochondria. The presence of mitochondrial material inside a vacuole is indicative of a compartment representing a bona fide autophagosome. Irgm1- induced vacuoles were positive for Mitotracker Red (MT) , with a sevenfold increas

47、e in MT+ cells among GFP-Irgm1transfected cells (65 5%) compared to controls (9.4 3.5%; P 0.01) . These characteristics are consistent with the interpretation that Irgm1- induced vacuoles were large autolysosomes.Irgm1-induced profiles were examined by electronmicroscopy. Vacuoles were not observed

48、in mock-transfected control cells (Fig. 1C, panel I; fig. S4) but were abundant in cells treated with IFN- (Fig. 1C, panel II, arrowheads). These compartments contained internal membranes. Similar types of vacuoles were observed in cells that were transfected with GFP-Irgm1 (Fig. 1C, panel III, arro

49、whead), consistent with autolysosomal nature of Irgm1-induced organelles. We also detected doublemembrane structures in GFPIrgm1 transfected cells (Fig. 1C, panels III and IV,arrows), a morphological signature of nascent autophagosomes, known as phagophores or isolation membranes. Thus, in addition

50、to large vacuoles representing autolysosomal structures,Irgm1 induces early autophagosomal organelles.Fig. S4. Enlarged version of Figure 1C.We next examined whether Irgm1 was necessary for early stages of the autophagosomal pathway. The formation of early auto-phagosomal precursors and newly comple

51、ted autophagosomes is quantifiable by following microtubuleassociated protein light-chain 3 (LC3) changes. LC3 (Atg8) exists in two forms: the cytosolic species LC3-I with an electrophoretic mobility corresponding to the relative molecular mass (Mr) of 18 kD, and its membrane-associated form, LC3-II

52、, conjugated C-terminally to phosphatidylethanolamine磷脂酰乙醇胺, with an apparent Mr of 16 kD. The latter form, LC3-II, inserts into the membrane of nascent autophagosomes and correlates with the appearance of LC3+autophago-somes . Increasing levels of LC3-II on immunoblots can be used to document induc

53、tion of autophagy. The intensity of the LC3-II band (Fig. 2, A and B) was increased in cells transfected with GFP-Irgm1 relative to the control cells transfected with GFP alone. The Irgm1-induced LC3-II levels were comparable to the effects of rapamycin, a conventional inducer of autophagy (Fig. 2,

54、A and B). Next, we tested whether Irgm1 was responsible for IFNinduced autophagy. For these studies, we reduced the expression of endogenous Irgm1 by small interfering RNA (siRNA) in IFN-stimulated cells, as confirmed by immunoblotting (Fig. 2C). Once lipidated, LC3 undergoes transition from the cyt

55、osolic form to a membranebound form, which is then scored as cytoplasmic puncta by fluorescence microscopy . RAW 264.7 cells were cotransfected with GFP-LC3 and Irgm1 siRNA or control scrambled siRNA, and the formation of GFP-LC3 puncta was analyzed upon stimulation with IFN-. Fig. 2. Irgm1 is requi

56、red for IFN-induced autophagy and transfer of mycobacteria from immature phagosomes to compartments with late endosomal/lysosomal characteristics.(A) RAW 264.7 cells were transiently transfected either with GFP and incubated with or without rapamycin (50 mg/ml) for 4 hours or transfected with GFP-Ir

57、gm1. Immunoblot analysis of cells was carried out with antibody to LC3 (anti-LC3).(B) Quantification of the LC3 band intensity.(C) RAW 264.7 cells were transfected with Irgm1 siRNA or control siRNA and incubated for 24 hours with or without IFN-g (200 U/ml). Cells were processed for immunoblotting a

58、nd probed for Irgm1. (D and E) Cells were transiently cotransfected with enhanced GFP(EGFP)LC3 and control siRNA or Irgm1 siRNA and treated with IFN-(200 U/ml) for 24 hours, and the number of GFP-LC3 puncta per cell was quantified. Results are shown as the meansSEM; *P 0.01 (ANOVA); n = 100 cells fr

59、om three independent experiments.(F) Knockdown of Beclin1 by siRNA examined by immunoblotting with anti-Beclin. (G and H) RAW 264.7 cells were transfected with GFP or cotransfected with GFP-Irgm1 and control siRNA or siRNA against Beclin 1. Cells were infected with Texas Redlabeled M. tuberculosis v

60、ar. bovis BCG for 15 min and chased for 1 hour. Phagosomal maturation was assessed by colocalization of BCG phagosomes (red) with lysosomal marker CD63 (blue) with anti-CD63 followed by incubation with secondary antibody conjugated to Alexa Fluor 647.(G) Main panels show merged three-color fluoresce