Colin Adrain, G.B

7/28/2019 Colin Adrain, G.B.

1/5

interacting partners in various species are important

steps towards understanding the control of this crucial

process. The contribution of Dcp1 and Hedls to Dcp2

activation can now be explored at the molecular level.

Further cellular, structural and enzymatic analyses will

undoubtedly reveal new interacting partners of the

decapping complex and enable the mechanism that leads

to the irrevocable decision to activate decapping to

be deciphered.

AcknowledgementsWe thank Andrzej Dziembowski for his help with the PyMOL software

and members of the Seraphin laboratory for their support. This work was

supported by La Ligue contre le Cancer (Equipe Labellisee 2005), the

CNRS and the Ministry of Research (ACI BCMS).

References1 Meyer, S. et al. (2004) Messenger RNA turnover in eukaryotes:

pathways and enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 197216

2 Coller, J. and Parker, R. (2004) Eukaryotic mRNA decapping. Annu.

Rev. Biochem. 73, 861890

3 Cougot, N. et al. (2004) Cap-tabolism. Trends Biochem. Sci. 29,

436444

4 Stoecklin, G. et al. (2006) ARE-mRNA degradation requires the 50

30

decay pathway. EMBO Rep. 7, 7277

5 Yamashita, A. et al. (2005) Concerted action of poly(A) nucleases and

decapping enzyme in mammalian mRNA turnover. Nat. Struct. Mol.

Biol. 12, 10541063

6 Lejeune, F. et al. (2003) Nonsense-mediated mRNA decay in

mammalian cells involves decapping, deadenylating, and exonucleo-

lytic activities. Mol. Cell 12, 675687

7 Conti, E. and Izaurralde, E. (2005) Nonsense-mediated mRNA decay:

molecular insights and mechanistic variations across species. Curr.

Opin. Cell Biol. 17, 316325

8 van Dijk, E. et al. (2002) Human Dcp2: a catalytically active mRNA

decapping enzyme located in specific cytoplasmic structures.EMBO J.

21, 69156924

9 Wang, Z. et al. (2002) The hDcp2 protein is a mammalian mRNA

decapping enzyme. Proc. Natl. Acad. Sci. U. S. A. 99, 1266312668

10 Lykke-Andersen, J. (2002) Identification of a human decapping

complex associated with hUpf proteins in nonsense-mediated decay.

Mol. Cell. Biol. 22, 81148121

11 Ingelfinger, D. et al. (2002) The human LSm1-7 proteins colocalize

with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct

cytoplasmic foci. RNA 8, 14891501

12 Eystathioy, T. etal. (2003) The GW182 protein colocalizes with mRNA

degradation associated proteins hDcp1and hLSm4 in cytoplasmicGW

bodies. RNA 9, 11711173

13 Sheth, U. and Parker, R. (2003) Decapping and decay of messengerRNA occur in cytoplasmic processing bodies. Science 300, 805808

14 Cougot, N. et al. (2004) Cytoplasmic foci are sites of mRNA decay in

human cells. J. Cell Biol. 165, 3140

15 She, M. et al. (2006) Crystal structure and functional analysis of

Dcp2p from Schizosaccharomyces pombe. Nat. Struct. Mol. Biol. 13,

6370

16 Fenger-Grn, M.et al. (2005) Multiple processing body factors and the

ARE binding protein TTP activate mRNA decapping. Mol. Cell 20,

905915

17 Steiger, M. et al. (2003) Analysis of recombinant yeast decapping

enzyme. RNA 9, 231238

18 Beelman, C.A. et al. (1996) An essential component of the decapping

enzyme required for normal rates of mRNA turnover. Nature 382,

642646

19 Dunckley, T. and Parker, R. (1999) The DCP2 protein is required for

mRNA decapping in Saccharomyces cerevisiae and contains afunctional MutT motif. EMBO J. 18, 54115422

20 She, M. et al. (2004) Crystal structure of Dcp1p and its functional

implications in mRNA decapping. Nat. Struct. Mol. Biol. 11, 249256

21 Coller, J.M. et al. (2001) The DEAD box helicase, Dhh1p, functions in

mRNA decapping and interacts with both the decapping and dead-

enylase complexes. RNA 7, 17171727

22 Badis, G. et al. (2004) Targeted mRNA degradation by deadenylation-

independent decapping. Mol. Cell 15, 515

23 Yu, J.H. et al. (2005) Ge-1 is a central component of the mammalian

cytoplasmic mRNA processing body. RNA 11, 17951802

0968-0004/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tibs.2006.03.001

Apoptosomes: protease activation platformsto die from

Colin Adrain, Gabriela Brumatti and Seamus J. Martin

Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland

Apoptosis is orchestrated by members of the caspase

family of cysteine proteases that exist as latent pro-

enzymes in healthy cells. Caspase-activating platforms,

called apoptosomes, initiate caspase activation in

metazoans as diverse as nematodes and mammals.

Several recent studies have generated new insights into

the composition and assembly mechanisms of worm, fly

and human apoptosomes.

Apoptosomes promote caspase activation

During apoptosis, cells actively participate in their own

elimination by activating proteolytic enzymes, called

caspases (for cysteine aspartic acid-specific proteases)

[1]. Initiator caspases become activated at the onset of

apoptosis and unleash a barrage of proteolytic activity

within the cell by activating downstream (effector)

caspases through direct proteolysis. Effector caspases

dismantle cellular structures and shut down vital bio-

chemical processes, resulting in controlled cell death and

phagocyte-mediated removal of cell corpses [2,3]. AnCorresponding author: Martin, S.J. ([email protected]).

Available online 3 April 2006

Update TRENDS in Biochemical Sciences Vol.31 No.5 May 2006 243

www.sciencedirect.com
mailto:[email protected]://www.sciencedirect.com/http://www.sciencedirect.com/mailto:[email protected]


2/5

important route to caspase activation involves the

assembly of caspase-activating complexes, called apopto-

somes, in response to diverse forms of cellular stress [4].

Three recent studies provide fresh insights into the

composition of worm, fly and mammalian apoptosomes

and highlight interesting differences between their

symmetry and assembly mechanisms [57].

Worm apoptosomes contain CED-4 tetramers

A single caspase, CED (cell death abnormal)-3, is both

necessary and sufficient for all developmental pro-

grammed cell deaths that take place in the nematode

C. elegans. Genetic and molecular studies have established

that CED-3 is activated in response to developmental cues

and that this requires its adapterprotein, CED-4. Normally,

CED-4 is restrained from activating CED-3 through

interaction with mitochondrion-tethered CED-9 [8,9].

CED-9 can be neutralized by EGL-1 (egg-laying defective-

1), which displaces it from CED-4 to trigger assembly of the

CED-4CED-3 apoptosome [9]. Until now, the structure of

the worm apoptosome was obscure.

Shi and colleagues [5] have recently reconstituted the

C. elegans cell-death pathway in vitro using purified

proteins and have found that CED-4, in its inactive state,

is an asymmetric dimer in complex with a single molecule

of CED-9 (Figure 1a). Similar observations have also been

made by Colman and colleagues [10], although these

authors conclude that the inactive CED-4CED-9 complex

possesses 2:2 stoichiometry. Assembly of the worm

apoptosome seems to require CED-4 to further oligomerize

into a tetrameric particle that functions as an activation

platform for CED-3 [5]. This is achieved through

TiBS

CED-9 CED-4

CED-3

DARK

DRONC

DIAP1

Inactive Active

(a) Worm

(b) Fly

(c) Mammalian

Apaf-1

Caspase-9

DRONC

Caspase-9

CED-3

Cyt cdATP

dATP

ATP

CED-4

DARK

Apaf-1

EGL-1CED-13

ReaperHid

GrimSickle

BH3-onlyproteins

Figure 1. Assembly of worm, fly and human apoptosomes. (a) In the nematode worm, CED-4 (grey octagon) and CED-3 (red circle) can be induced to form apoptosomes

throughdisplacement of the cell-death-inhibitory protein,CED-9(purple), fromCED-4 dimers.CED-9 canbe displaced by either of theBH3-onlyproteins EGL-1 or CED-13,and

thispermitsthe further dimerizationof CED-4dimersto formtetramers thatcan activatethe CED-3 caspase. (b) In the fly, the CED-4-related protein,DARK (grey octagon),can

oligomerize into an octameric structure that contains the CED-3-related protease DRONC (red circle). Fly apoptosomes are thought to be prevented from assemblingthrough

inhibition of DRONC by DIAP1. DIAP1 can be neutralized by the small proteins Reaper, Hid, Grim or Sickle (which are induced in response to specific stimuli) and this permits

assembly of the octameric fly apoptosome. (c) In mammals, the CED-4-related protein Apaf-1 can be induced to oligomerize into heptameric apoptosomes that contain

caspase-9. Thisis controlled by a family of proteins(the BH3-onlymembers of theBcl-2 family) thatbecomeactivated in responseto death triggersand liberateCyt cfromthe

mitochondrial intermembrane space. Cyt cacts as a co-factor for apoptosome assembly and, in association with dATP, triggers oligomerization of Apaf-1 and its association

with caspase-9.

Update TRENDS in Biochemical Sciences Vol.31 No.5 May 2006244

http://www.sciencedirect.com/http://www.sciencedirect.com/


3/5

displacing CED-9 from the CED-4 dimer by the Bcl-2

homology 3 (BH3)-only protein, EGL-1 (or the related

CED-13), which induces a major conformational change in

CED-9 and disrupts the binding interface between the

latter and CED-4 [5]. The number of molecules of CED-3

that become associated with the tetrameric CED-4

apoptosome remains unclear, but this could be as many

as eight if each CED-4 molecule recruits a CED-3

homodimer into the complex (Figure 1a). Further studiesare needed to answer this question and also to provide a

high-resolution view of the fully armed CED-4CED-

3 apoptosome.

Fly apoptosomes contain octamers of DARK

In Drosophila, the CED-4 relative DARK (Drosophila

Apaf-1-related killer) is essential for programmed cell

death in many situations and this protein selectively

interacts with the fly caspase DRONC (Drosophila Nedd2-

like caspase) [11,12]. Although similar in domain organ-

ization to CED-4, DARK is a substantially larger protein

owing to the presence of multiple WD40 repeats at its C

terminus; this region within DARK might have an auto-

inhibitory function. Curiously, DRONC activation by

DARK does not seem to be regulated by either of the two

known fly relatives of CED-9. Indeed, it is not clear what

role, if any, CED-9-related proteins have in the regulation

of programmed cell death in the fly because data have yet

to emerge to suggest that they function as important

arbiters of cell fate in this organism. Instead, the fly death

machinery is repressed mainly by the caspase-inhibitory

protein Drosophila inhibitor-of-apoptosis protein 1

(DIAP1) [13]. Inactivation of the gene encoding DIAP1

results in widespread cell death, leading to lethality early

in development. Thus, DIAP1 might constitutively associ-

ate with fly caspases such as DRONC and prevent

spontaneous activation of these proteases (Figure 1b).

Akey and colleag ues have recent ly res olve d the

structure of oligomerized DARK at 18.8 A using electron

cryo-microscopy [6]. The active DARK apoptosome is an

impressive wheel-like octamer that forms in the presence

(a) Fly

Top view Bottom view Domain model

(b) Human

Top view Bottom view Domain model

CARD NBD HD1 WHD HD2 8 6

CARD Cyt cNBD HD1 WHD HD2 7 6

Figure 2. Structure of the fly and human apoptosomes. (a) Surface views (left and middle) and domain model (right) of the fly apoptosome. The fly apoptosome comprises

eight molecules of DARK (depicted) and an, as yet, unknown number of molecules of the fly caspase DRONC (not shown). Assembly of the fly apoptosome from DARK

monomers can be initiated through neutralization of the fly caspase-inhibitory protein, DIAP1, by Reaper, Hid or Grim. Assembled apoptosomes are likely to recruit several

molecules of the DRONC protease (via the CARD domains of DARK that are located towards the centre of the apoptosome), which probably results in DRONC activation

through an allosteric mechanism. (b) Surface views (left and middle) and domain model (right) of the human apoptosome. Assembly of Apaf-1 apoptosomes is initiated by

theescapeof Cyt cfromthe mitochondrial intermembrane spaceinto the cytosol.This event is regulated by members of the Bcl-2familythat caneither promoteCyt crelease

(BH3-only proteinsand the Baxsubfamily)or antagonizeit (Bcl-2, Bcl-xL andrelatedproteins).Cyt cpromotesApaf-1oligomerizationby interacting withthe C-terminal WD40

repeats within Apaf-1, which fold into two b-propellers that are located towards the periphery of the structure. Assembled Apaf-1 apoptosomes recruit several molecules of

caspase-9 (not shown) and promote auto-activation of this protease through an allosteric mechanism. Domains within DARK and Apaf-1 are colour-coded as indicated.

Abbreviations: b, b propellers; CARD, caspase-recruitment domain; HD, helix domain; NBD, nucleotide-binding domain; WHD, winged-helix domain. Part (a) reproduced,

with permission, from Ref. [6]; Part (b) reproduced, with permission, from Ref. [7].




4/5

of dATP (2 0-deoxyadenosine 50-triphosphate), but not ATP

(Figures 1b and 2a). Interestingly, DARK apoptosomes

were also found to further dimerize into double-wheeled

structures, but the significance of this remains unclear at

present. The number of DRONC molecules that become

recruited to DARK apoptosomes is also not known because

these were assembled in the absence of caspase, but it is

likely that it is a similar number to that of DARK

subunits. The natural trigger for oligomerization ofDARK is not yet clear and it remains possible that binding

partners that repress spontaneous DARK oligomerization

in vivo exist. It is also possible that DARK could exist in a

pre-oligomerized state and that activation of the fly

apoptosome is regulated at the level of DRONC recruit-

ment into the complex. Activation of the fly apoptosome is

controlled by three proteins Reaper, Hid and Grim the

expression of which can be induced by various death

stimuli [13,14]. Any one of these three proteins can

neutralize DIAP1 and can either permit recruitment of

DRONC into the apoptosome or unmask the activity of

pre-recruited DRONC. The structure of the fully

assembled fly apoptosome loaded with the DRONCprotease awaits determination.

Human apoptosomes contain Apaf-1 heptamers

In vertebrates, the CED-4-relative Apaf-1 constitutes the

scaffold upon which the apoptosome is assembled [15].

Like DARK, the C-terminal region of Apaf-1 contains

multiple WD40 repeats, which are absent in CED-4, and

these are involved in regulating the assembly of the Apaf-

1 apoptosome. Although 12 human caspases have been

identified, only one of these caspase-9 can associate

with Apaf-1 to form apoptosomes [15,16]. Surprisingly,

assembly of the human apoptosome is controlled by

the electron-transport protein cytochrome c (Cyt c),

which is released from mitochondria during apoptosis

and triggers oligomerization of Apaf-1 into a wheel-like

heptameric complex (Figures 1c and 2b). Although human

relatives of CED-9 including Bcl-2 and Bcl-xL do

regulate the assembly of Apaf-1 apoptosomes, this is

achieved indirectly by regulating the release of Cyt c from

the mitochondrial intermembrane space and not through

direct interaction with Apaf-1 [17].

A recent paper from Akey and colleagues has provided

new views of the Apaf-1 apoptosome at 12.8 A resolution

[7]. The human and fly apoptosomes are similar in overall

structure, but a major difference is the number of subunits

they contain (Figure 2). The central hub of the Apaf-1

apoptosome is formed by lateral interactions between thenucleotide-binding and oligomerization domains. The hub

is capped with a crown of caspase-recruitment domains

(CARDs), which are the regions within Apaf-1 that are

responsible for capturing caspase-9 molecules through a

similar motif located at the N terminus of caspase-9.

The spokes of the apoptosome are formed by the

C-terminal WD-40 repeats, which fold into two b

propellers to form a cleft that is likely to be important in

regulating the propensity of Apaf-1 to oligomerize. To

date, the precise location and number of Cyt c molecules

within the human apoptosome has been obscure, although

it has been proposed that Cyt c binding to the cleft within

the Apaf-1 b propellers could trigger a conformational

change that permits apoptosome oligomerization [18,19].

Confirming this, Akey and colleagues docked a single

molecule of Cyt c within this cleft, suggesting that Cyt c

binds to Apaf-1 with a 1:1 stoichiometry [7]. However, it

should be noted that the assignments of the b-propellers

and Cyt c are based on docking experiments rather than

genuine atomic structures. The current study by Akey and

colleagues provides a strikingly detailed view of theassembled apoptosome, albeit lacking Caspase-9 [7], but,

from previous studies, it is likely that the caspase forms a

dome on top of the central hub [19] (Figure 2). However,

the precise arrangement and number of caspase-9

molecules within the Apaf-1 apoptosome remains to be

determined. Consequently, it is also unclear how recruit-

ment of caspase-9 to the apoptosome results in activation

of this protease, although an allosteric mechanism

is probable.

Structural clues about apoptosome latency

Alth ough assembl y of the worm, fly and human

apoptosomes is regulated in different ways, they sharea common requirement for binding of ATP or dATP and

an ability to form wheel-like oligomeric structures.

Although there is no evidence at present that either

CED-4 or DARK can hydrolyse the bound nucleotide, a

recent study from Wang and colleagues indicates that

dATP hydrolysis is essential for proper assembly of the

Apaf-1 apoptosome [20]. In the absence of Cyt c, Apaf-1

is monomeric, contains a single dATP molecule and

cannot interact with Caspase-9. The new data suggest

that Cyt c triggers a conformational change within Apaf-

1 that is driven by hydrolysis of the bound dATP to

dADP, followed by exchange for dATP and oligomeriza-

tion of Apaf-1 into the heptameric complex that can then

recruit and activate caspase-9 [7,20].

Concluding remarks

Recent studies have provided substantial insights into the

structure of worm, fly and human apoptosomes but it

should be noted that, in all cases, these apoptosomes have

been built using recombinant proteins in the absence of

their caspase partners. The isolation and characterization

of native apoptosomes from apoptotic cells is likely to be a

formidable challenge. In addition, several important

questions remain unresolved. Although, it is clear that

CED-3 is an integral part of the worm apoptosome, few

substrates for this protease have yet to be identified. A

similar lack of knowledge pertains to how DRONCcoordinates death in the fly, although, in this instance,

at least two other proteases are activated downstream. It

is also perplexing that, although CED-9 relatives have

been found in the fly, they do not seem to have either a

direct or indirect role in regulating assembly of the fly

apoptosome. Neither does there seem to be a role for Cyt c

in regulating fly apoptosomes, despite the structural

similarity between Apaf-1 and DARK. In mammals, the

Apaf-1caspase-9 apoptosome is now well characterized,

but several studies have provided tantalizing evidence

that another apoptosome that activates caspase-2 could

also exist [2123].

Update TRENDS in Biochemical Sciences Vol.31 No.5 May 2006246



5/5

To die will be an awfully big adventure (J.M. Barrie)

how true this has turned out to be, at least at the

cellular level.

AcknowledgementsWe apologize to authors whose papers could not be cited owing to space

constraints. We are grateful to Science Foundation Ireland for ongoing

support of work in our laboratory and to Christopher W. Akey for kindly

providing the structures presented in Figure 2.

References1 Fuentes-Prior, P. and Salvesen, G.S. (2004) The protein structures

that shape caspase activity, specificity, activation and inhibition.

Biochem. J. 384, 201232

2 Fischer, U. et al. (2003) Many cuts to ruin: a comprehensive update of

caspase substrates. Cell Death Differ. 10, 76100

3 Savill, J. and Fadok, V. (2000) Corpse clearance defines the meaning of

cell death. Nature 407, 784788

4 Adrain, C. and Martin, S.J. (2001) The mitochondrial apoptosome: a

killer unleashed by the cytochrome seas. Trends Biochem. Sci. 26,

390397

5 Yan, N. et al. (2005) Structure of the CED-4CED-9 complex provides

insights into programmed cell death in Caenorhabditis elegans.

Nature 437, 831837

6 Yu, X. et al. (2006) Three-dimensional structure of a doubleapoptosome formed by the Drosophila Apaf-1 related killer. J. Mol.

Biol. 355, 577589

7 Yu, X. et al. (2005) Structure of the human apoptosome at 12.8 A

resolution provides insights into this cell death platform.Structure 13,

17251735

8 Spector, M.S. et al. (1997) Interaction between the C. elegans cell-

death regulators CED-9 and CED-4. Nature 385, 653656

9 Chen, F. et al. (2000) Translocation of C. elegans CED-4 to nuclear

membranes during programmed cell death. Science 287,

14851489

10 Fairlie, W.D.et al. (2006) CED-4 forms a 2:2 heterotetrameric complex

with CED-9 until specifically displaced by EGL-1 or CED-13. Cell

Death Differ. 13, 426434

11 Abrams, J.M. (1999) An emerging blueprint for apoptosis in

Drosophila. Trends Cell Biol. 9, 435440

12 Quinn, L.M. et al. (2000) An essential role for the caspase DRONC in

developmentally programmed cell death inDrosophila.J. Biol. Chem.

275, 4041640424

13 Wang, S.L. et al. (1999) The Drosophila caspase inhibitor DIAP1 is

essential for cell survival and is negatively regulated by HID. Cell 98,

453463

14 Goyal, L. et al. (2000) Induction of apoptosis by Drosophila Reaper,

Hid and Grim through inhibition of IAP function. EMBO J. 19,

589597

15 Li, P. et al. (1997) Cytochrome c and dATP-dependent formation of

Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell

91, 479489

16 Hill, M.M. et al. (2004) Analysis of the composition, assembly

kinetics and activity of native Apaf-1 apoptosomes. EMBO J. 23,

21342145

17 Newmeyer, D.D. and Ferguson-Miller, S. (2003) Mitochondria:

releasing power for life and unleashing the machineries of death.

Cell 112, 481490

18 Adrain, C. et al. (1999) Regulation of apoptotic protease activating

factor-1 oligomerization and apoptosis by the WD-40 repeat region.

J. Biol. Chem. 274, 2085520860

19 Acehan, D. et al. (2002) Three-dimensional structure of the

apoptosome: implications for assembly, procaspase-9 binding, and

activation. Mol. Cell 9, 423432

20 Kim, H.E. et al. (2005) Formation of apoptosome is initiated by

cytochrome c-induced dATP hydrolysis and subsequent nucleotide

exchange on Apaf-1. Proc. Natl. Acad. Sci. U. S. A. 102, 1754517550

21 Read, S.H. et al. (2002) A novel Apaf-1-independent putative caspase-

2 activation complex. J. Cell Biol. 159, 739745

22 Tinel, A. and Tschopp, J. (2004) The PIDDosome, a protein complex

implicated in activation of caspase-2 in response to genotoxic stress.

Science 304, 843846

23 Tu, S. et al. (2006) In situ trapping of activated initiator caspases

reveals a role for caspase-2 in heat shock-induced apoptosis. Nat. Cell

Biol. 8, 7277

0968-0004/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tibs.2006.03.004

Assembling the bacterial segrosome

Finbarr Hayes1 and Daniela Barilla` 2

1Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street,

Manchester, M1 7ND, UK2Department of Biology (Area 10), University of York, PO Box 373, York, YO10 5YW, UK

Genome segregation in prokaryotes is a highly orderedprocess that integrates with DNA replication, cytokin-

esis and other fundamental facets of the bacterial cell

cycle. The segrosome is the nucleoprotein complex that

mediates DNA segregation in bacteria, its assembly and

organization is best understood for plasmid partition.

The recent elucidation of structures of the ParB plasmid

segregation protein bound to centromeric DNA, and of

the tertiary structures of other segregation proteins, are

key milestones in the path to deciphering the molecularbasis of bacterial DNA segregation.

DNA segregation in prokaryotes

DNA segregation is a fundamental process that cells

perform with high precision to ensure stable genome

transmission during cytokinesis. Whereas chromosome

segregation in eukaryotes is comparatively well described

[1], the understanding of the molecular mechanisms that

direct genome partition in prokaryotes is more rudimen-

tary. Plasmids are tractable model systems with which to

decipher the DNA-segregation process in bacteria:

Corresponding authors: Hayes, F. ([email protected]), Barilla, D.

([email protected] ).

Available online 11 April 2006


http://-/?-mailto:[email protected]:[email protected]:[email protected]://www.sciencedirect.com/http://www.sciencedirect.com/mailto:[email protected]:[email protected]://-/?-

Documents

Colin Adrain, G.B