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190 Part II Cellular Basis of Hematology

NONAPOPTOTIC ROLES FOR CASPASES to form the “apoptosome,” which in turn triggers downstream effec-
tor caspases (see Fig. 18.5B). Other apoptogenic factors released from
Although justifiably known for their apoptotic functions, there is mitochondria, including apoptosis-inducing factor (AIF), SMAC/
7
accumulating evidence that caspases also function in healthy cells. Diablo, Omi/HtrA2, and endonuclease G, augment apoptosis.
Caspase-1 was originally identified as the processing enzyme for
interleukin 1β (IL-1β), and subsequently shown to process another
proinflammatory cytokine IL-18. Caspases can also be involved in BCL-2 FAMILY PROTEINS AND THE INTRINSIC
negative-feedback control of erythroblast differentiation by mature PATHWAY OF APOPTOSIS
erythroblasts through degradation of GATA-1. Several dramatic
structural alterations associated with cell differentiation also appear The BCL-2 family of proteins constitutes a critical control point
to require transient caspase activation. Cleavage of a limited number in apoptosis residing immediately upstream to irreversible cellular
of caspase substrates precede nuclear and chromatin changes during damage, where the members control MOMP. 9–13 Several BCL-2
terminal erythroid differentiation, and caspase inhibitors block proteins reside at subcellular membranes, including the mitochon-
proplatelet formation from megakaryocyte and macrophage differ- drial outer membrane, the endoplasmic reticulum (ER), and nuclear
entiation. Caspase-8, in some contexts, has a prosurvival function, membranes. The different anti- and proapoptotic members of this
inhibiting necroptosis or apoptosis. The more limited caspase activa- family form a highly selective network of functional interactions
tion in these instances may involve some degree of compartmentaliza- that ultimately governs MOMP. The founding member of this
tion in space or time. Since the activity of unprocessed apical caspases family, BCL-2, was discovered as the defining oncogene in follicular
requires persistent binding to adaptor proteins, this constraint may lymphomas, located at one reciprocal breakpoint of the t(14;18)
allow for localized, limited caspase activity under some circumstances (q32;q21) chromosomal translocation. Cells transduced with BCL-2
consistent with a nonapoptotic role. remained viable for extended periods in the absence of growth factors.
Transgenic mice bearing a BCL-2-Ig mini-gene recapitulating the
t(14:18) chromosomal translocation displayed B-cell follicular hyper-
INHIBITORS OF APOPTOSIS PROTEINS plasia and progressed over time to diffuse large B-cell lymphomas.
BCL-2 expression specifically blocked the morphologic features of
The only known endogenous caspase inhibitor in mammalian cells is apoptosis, including plasma membrane blebbing, nuclear condensa-
a member of the inhibitor of apoptosis proteins (IAPs) family. IAPs tion, and DNA cleavage. Importantly, unlike other oncogenes known
were originally described in insect viruses as viral proteins produced at that time, BCL-2 did not promote proliferation, defining a new
10
8
during cellular infection to block host cell apoptosis. In mamma- category of oncogenes, namely regulators of cell death. The first
lian cells, X-linked inhibitor of apoptosis (XIAP) is the only fully proapoptotic BCL-2 homologous protein to be identified, BAX,
validated caspase inhibitor. XIAP binds to the active sites of specific coimmunoprecipitated in stoichiometric amounts with BCL-2.
4,8
caspases to block catalytic activity or interferes with dimerization BAX-transfected cells died rapidly in the absence of growth factor
(caspase-9). IAPs contain one to three baculovirus IAP repeat (BIR) and BAX was subsequently shown to be capable of directly trigger-
domains that coordinate zinc, and one or more additional protein- ing apoptosis. Since the discovery of BCL-2 and BAX, the BCL-2
interaction domains. IAP-binding motifs (IBMs) consist of a short family in mammals has expanded, with several family members acting
peptide sequence with an N-terminal alanine and bind to a surface principally as prosurvival proteins and others hastening cell death
groove on certain BIR domains. Initial processing of caspase-3, -7, in various experimental systems (Fig. 18.6). Homologs of BCL-2
and -9 generates an IBM at the N-terminal end of the short subunit, proteins exist in all metazoans studied to date, as well as several
providing an anchor point for additional physical interactions with animal DNA viruses.
IAP proteins. XIAP uses different BIR domains to bind IBMs of The ability of BCL-2 family proteins to selectively bind each other
specific caspases. is integral to their function in regulating MOMP and apoptosis. These
Two proteins normally localized in the mitochondrial inter- interactions are modulated by conserved homology domains (BH),
membrane space, second mitochondria-derived activator of caspase which correspond to α-helical and connecting segments that dictate
(SMAC)/Diablo and Omi/HtrA2, can bind IAPs via an NH 2 - structure and function (see Fig. 18.6). 9–13 All antiapoptotic members,
terminal IBM sequence and competitively displace bound caspases. such as BCL-2 and BCL-X L , and a subset of proapoptotic family
Whereas the NH 2 -terminus of active SMAC/Diablo is generated by members, such as BAX and BAK, are “multidomain” proteins sharing
removal of a presequence during mitochondrial import, Omi/HtrA2 sequence homology within 3–4 BH domains. The “BH3-only” subset
is a stress-activated serine protease that is cleaved by autoprocessing. of proapoptotic molecules, including BCL-2 antagonist of cell death
Cytoplasmic translocation of SMAC/Diablo and Omi/HtrA2 during (BAD), BID, BCL-2 interacting mediator of cell death (BIM), NOXA,
apoptosis provides an additional mechanism for caspase activation. and p53 upregulated modulator of apoptosis (PUMA), show sequence
homology only within a single α-helical segment, the BH3 domain,
which is also known as the critical death domain required for binding
CORE APOPTOSIS PATHWAYS to “multidomain” BCL-2 family members. BCL-2 family interactions
ultimately regulate mitochondrial intramembranous oligomerization
In mammals, the execution of apoptosis downstream of death signals of BAX/BAK, which is the prime mechanism of MOMP. BAX and
is governed by two molecular programs that terminate in caspase BAK are absolutely required to execute death by all apoptotic signals
activation, which may be linked in certain cell types. The extrinsic that activate the intrinsic pathway, nominating these molecules as the
pathway operates downstream of death receptors, such as Fas and requisite gateway to the mitochondrial apoptotic machinery.
other members of the TNF receptor family, which recruit DISC upon A combination of genetic, biochemical, and structural studies has
ligand binding. This complex, in turn, recruits and activates caspase-8 begun to unravel the molecular mechanism underlying regulation
and -10, leading to activation of other downstream caspases. The of MOMP by BCL-2 proteins (Fig. 18.7). BH3-only molecules
second program, also known as the intrinsic pathway, is marked by the are upstream sentinels that selectively respond to proximal death
involvement of mitochondria. 9–12 Besides their role in biosynthesis, and survival signals to regulate BAX/BAK oligomerization. 9–12
calcium buffering, and ATP production, mitochondria participate in However, their apoptotic activity is suppressed unless activated by
apoptosis by releasing factors such as cytochrome c, a component of transcriptional and posttranslational mechanisms in a tissue- and
the mitochondrial electron transport chain. The permeabilization of signal-specific manner. 10,12 For example, the activation of NOXA and
the outer mitochondrial membrane (MOMP) and release of apopto- PUMA is under direct transcriptional regulation by p53, a finding
genic factors marks the “point of no return” in the intrinsic pathway that is consistent with their roles as specialized death sentinels during
of apoptosis, and is exquisitely regulated by BCL-2 family proteins. DNA damage. On the other hand, cytosolic BID is activated upon
Once released, cytochrome c is assembled with APAF-1 and caspase-9 cleavage by caspase-8, and the apoptotic activity of BAD is regulated