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Biomedical subjects

Catherine L Day

Publications and source records attributed to Catherine L Day.

13 recordsLinked to original sources
PubMed2006

The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized.

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.

Animals↗
PubMed2005

Regulation of apoptosis: uncovering the binding determinants.

Eukaryotic cells use complex networks of signal transduction proteins to make decisions about whether to differentiate, grow or die. In the case of apoptosis, which is responsible for the programmed death of unwanted or damaged cells in multicellular organisms, recent structural, biochemical and cell-based assays have enhanced our understanding of the mechanisms by which some of the key proteins regulate this process. These studies have highlighted a critical role for conformational change and the regulated formation of specific complexes that can either inhibit or stimulate apoptosis. In some cases, it is still not clear what distinguishes inhibitory from activating complexes, but the value of a structural understanding is highlighted by the success of recent structure-based drug discovery programs that have targeted these complexes.

Animals↗
PubMed2005

Determination of cell survival by RING-mediated regulation of inhibitor of apoptosis (IAP) protein abundance.

Inhibitor of apoptosis (IAP) proteins, which bind to caspases via their baculoviral IAP repeat domains, also bear RING domains that enable them to promote ubiquitylation of themselves and other interacting proteins. Here we show that the RING domain of cIAP1 allows it to bind directly to the RING of X-linked IAP, causing its ubiquitylation and degradation by the proteasome, thus revealing a mechanism by which IAPs can regulate their abundance. Expression of a construct containing the RING of cellular IAP1 was able to deplete melanoma cells of endogenous X-linked IAP, promoted apoptosis, and also markedly reduced their clonogenicity when treated with cisplatin. Cross control of protein levels by RING domains may therefore enable their levels to be manipulated therapeutically.

Amino Acid Sequence↗
PubMed2005

Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function.

Apoptosis is initiated when Bcl-2 and its prosurvival relatives are engaged by proapoptotic BH3-only proteins via interaction of its BH3 domain with a groove on the Bcl-2-like proteins. These interactions have been considered promiscuous, but our analysis of the affinity of eight BH3 peptides for five Bcl-2-like proteins has revealed that the interactions vary over 10,000-fold in affinity, and accordingly, only certain protein pairs associate inside cells. Bim and Puma potently engaged all the prosurvival proteins comparably. Bad, however, bound tightly to Bcl-2, Bcl-xL, and Bcl-w but only weakly to A1 and not to Mcl-1. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. The results suggest that apoptosis relies on selective interactions between particular subsets of these proteins and that it should be feasible to discover BH3-mimetic drugs that inactivate specific prosurvival targets.

Amino Acid Sequence↗
PubMed2004

Solution structure of prosurvival Mcl-1 and characterization of its binding by proapoptotic BH3-only ligands.

The B cell lymphoma-2 (Bcl-2) homologs myeloid cell leukemia-1 (Mcl-1) and A1 are prosurvival factors that selectively bind a subset of proapoptotic Bcl homology (BH) 3-only proteins. To investigate the molecular basis of the selectivity, we determined the solution structure of the C-terminal Bcl-2-like domain of Mcl-1. This domain shares features expected of a prosurvival Bcl-2 protein, having a helical fold centered on a core hydrophobic helix and a surface-exposed hydrophobic groove for binding its cognate partners. A number of residues in the binding groove differentiate Mcl-1 from its homologs, and in contrast to other Bcl-2 homologs, Mcl-1 has a binding groove in a conformation intermediate between the open structures characterized by peptide complexes and the closed state observed in unliganded structures. Mutagenesis of potential binding site residues was used to probe the contributions of groove residues to the binding properties of Mcl-1. Although mutations in Mcl-1 had little impact on binding, a single mutation in the BH3-only ligand Bad enabled it to bind both Mcl-1 and A1 while retaining its binding to Bcl-2, Bcl-xL, and Bcl-w. Elucidating the selective action of certain BH3-only ligands is required for delineating their mode of action and will aid the search for effective BH3-mimetic drugs.

Amino Acid Sequence↗
PubMed2004

Localization of dynein light chains 1 and 2 and their pro-apoptotic ligands.

The dynein and myosin V motor complexes are multi-protein structures that function to transport molecules and organelles within the cell. DLC (dynein light-chain) proteins, found as components of both dynein and myosin V motor complexes, connect the complexes to their cargoes. One of the roles of these motor complexes is to selectively sequester the pro-apoptotic 'BH3-only' (Bcl-2 homology 3-only) proteins, Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor), and so regulate their cell death-inducing function. In vivo DLC2 is found exclusively as a component of the myosin V motor complex and Bmf binds DLC2 selectively. On the other hand, Bim interacts with DLC1 (LC8), an integral component of the dynein motor complex. The two DLCs share 93% sequence identity yet show unambiguous in vivo specificity for their respective BH3-only ligands. To investigate this specificity the three-dimensional solution structure of DLC2 was elucidated using NMR spectroscopy. In vitro structural and mutagenesis studies show that Bmf and Bim have identical binding characteristics to recombinant DLC2 or DLC1. Thus the selectivity shown by Bmf and Bim for binding DLC1 or DLC2, respectively, does not reside in their DLC-binding domains. Remarkably, mutational analysis of DLC1 and DLC2 indicates that a single surface residue (residue 41) determines the specific localization of DLCs with their respective motor complexes. These results suggest a molecular mechanism for the specific compartmentalization of DLCs and their pro-apoptotic cargoes and implicate other protein(s) in defining the specificity between the cargoes and the DLC proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity.

Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

Animals↗
PubMed2003

The structure of Bcl-w reveals a role for the C-terminal residues in modulating biological activity.

Pro-survival Bcl-2-related proteins, critical regulators of apoptosis, contain a hydrophobic groove targeted for binding by the BH3 domain of the pro-apoptotic BH3-only proteins. The solution structure of the pro-survival protein Bcl-w, presented here, reveals that the binding groove is not freely accessible as predicted by previous structures of pro-survival Bcl-2-like molecules. Unexpectedly, the groove appears to be occluded by the C-terminal residues. Binding and kinetic data suggest that the C-terminal residues of Bcl-w and Bcl-x(L) modulate pro-survival activity by regulating ligand access to the groove. Binding of the BH3-only proteins, critical for cell death initiation, is likely to displace the hydrophobic C-terminal region of Bcl-w and Bcl-x(L). Moreover, Bcl-w does not act only by sequestering the BH3-only proteins. There fore, pro-survival Bcl-2-like molecules probably control the activation of downstream effectors by a mechanism that remains to be elucidated.

Amino Acid Sequence↗
PubMed2002

Structure of a domain-opened mutant (R121D) of the human lactoferrin N-lobe refined from a merohedrally twinned crystal form.

Human lactoferrin is an iron-binding protein with a bilobal structure. Each lobe contains a high-affinity binding site for a single Fe(3+) ion and an associated CO(3)(2-) ion. Although iron binds very tightly, it can be released at low pH, with an accompanying conformational change in which the two domains move apart. The Arg121Asp (R121D) mutant of the N-lobe half-molecule of human lactoferrin was constructed in order to test whether the Asp121 side chain could substitute for the CO(3)(2-) ion at the iron-binding site. The R121D mutant protein was crystallized in its apo form as it lost iron during crystallization. The crystals were also merohedrally twinned, with a twin fraction close to 0.5. Starting from the initial molecular-replacement solution [Breyer et al. (1999), Acta Cryst. D55, 129-138], the structure has been refined at 3.0 A resolution to an R factor of 13.9% (R(free) of 19.9%). Despite the moderate resolution, the high solvent content and non-crystallographic symmetry contributed to electron-density maps of excellent quality. Weakened iron binding by the R121D mutant is explained by occlusion of the anion-binding site by the Asp side chain. The opening of the two domains in the apoR121D structure (a rotation of 54 degrees ) closely matches that of the N-lobe in full-length lactoferrin, showing that the extent of the conformational change depends on properties inherent to the N-lobe. Differences in the C-terminal portion of the N-lobe (residues 321-332) for apoR121D relative to the closed wild-type iron-bound structure point to the importance of this region in stabilizing the open form.

Amino Acid Substitution↗
PubMed2002

The anti-apoptotic activity of XIAP is retained upon mutation of both the caspase 3- and caspase 9-interacting sites.

The X-linked mammalian inhibitor of apoptosis protein (XIAP) has been shown to bind several partners. These partners include caspase 3, caspase 9, DIABLO/Smac, HtrA2/Omi, TAB1, the bone morphogenetic protein receptor, and a presumptive E2 ubiquitin-conjugating enzyme. In addition, we show here that XIAP can bind to itself. To determine which of these interactions are required for it to inhibit apoptosis, we generated point mutant XIAP proteins and correlated their ability to bind other proteins with their ability to inhibit apoptosis. partial differential RING point mutants of XIAP were as competent as their full-length counterparts in inhibiting apoptosis, although impaired in their ability to oligomerize with full-length XIAP. Triple point mutants, unable to bind caspase 9, caspase 3, and DIABLO/HtrA2/Omi, were completely ineffectual in inhibiting apoptosis. However, point mutants that had lost the ability to inhibit caspase 9 and caspase 3 but retained the ability to inhibit DIABLO were still able to inhibit apoptosis, demonstrating that IAP antagonism is required for apoptosis to proceed following UV irradiation.

Apoptosis↗
PubMed2002

HtrA--a renaissance protein.

Initially identified as proteases, members of the HtrA/DegP family of proteins have also been shown to act as chaperones in bacteria, and more recently implicated, as regulators of apoptosis in mammals. Two new structures of mammalian HtrA2 and E. coli DegP provide insights into the origin of this plurality of function.

Apoptosis↗
PubMed2001

HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins.

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.

Amino Acid Sequence↗