Completing the family portrait of the anti‐apoptotic Bcl‐2 proteins: Crystal structure of human Bfl‐1 in complex with Bim

Herman, Michael; Nyman, T.; Welin, M.; Lehtiö, L.; Flodin, S.; Tresaugues, L.; Kotenyova, T.; Flores, Alex F. C.; Nordlund, P.

doi:10.1016/j.febslet.2008.09.028

Cited by 68 publications

(83 citation statements)

References 33 publications

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“…The overall structure of Bcl-2 bound to the HBx BH3-like motif is similar to those reported for the Bcl-xL-Bad and Bcl-2A1-Bim complexes (32,34) (Fig. 4A).…”

Section: Resultssupporting

confidence: 79%

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

Jiang

Liu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 μM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.Bcl-2 | hepatitis B virus | apoptosis | crystal structure T he human hepatitis B virus (HBV) is a prototypical hepadnavirus that specifically infects hepatocytes (1, 2). Over 240 million people worldwide are chronically infected by HBV according to the World Health Organization. Persistent HBV infection is one of the major risk factors for the development of hepatocellular carcinoma (HCC), accounting for over 50% of all HCC cases (3, 4). The genome of HBV encodes only four proteins, among which the product of the X gene (HBx) is least understood (1).HBx contains 154 residues and is closely associated with HCC development. The X gene is most frequently integrated and preferentially maintained in HBV-associated HCC, and HBx expression is frequently detected in HCC patients (5, 6). Several studies using transgenic mice and cell culture models yielded contrasting conclusions. Some studies suggest that the development of HCC is closely linked to HBx expression (7-10), although HBx alone may be insufficient for HCC development (11,12). Other results suggest that HBx has the ability to suppress cell transformation through induction of apoptosis (13,14). As such, the molecular mechanism of HBx-induced cellular transformation remains enigmatic.Numerous cellular activities are reported to be modulated by HBx through interactions with various host targets. For example, HBx-mediated transcriptional activation is achieved through its interaction with transcription factors (15, 16) or components involved in the basal transcriptional machinery (17-19). HBx can also modulate cellular proliferation and viability through interactions with p53 and Bcl-2 family members. Reflecting its enigmatic functions, HBx has been demonstrated to either induce apoptosis (20) or prevent apoptosis (21).HBx was recently found to contain a BH3-like motif in its C-terminal sequences (22). Experiments carried out in human hepato...

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“…The overall structure of Bcl-2 bound to the HBx BH3-like motif is similar to those reported for the Bcl-xL-Bad and Bcl-2A1-Bim complexes (32,34) (Fig. 4A).…”

Section: Resultssupporting

confidence: 79%

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

Jiang

Liu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The BH1 and BH2 domains are conserved with the other pro-survival family members, with a less conserved BH3 domain, and a very limited homology in the BH4 domain 414 . Bfl-1 contains nine -helices which fold up to create a hydrophobic pocket similar to the one found in Bcl-xL where Bak binds 415 .…”

Section: The Structure Of Bfl-1mentioning

confidence: 99%

“…Bfl-1 contains nine -helices which fold up to create a hydrophobic pocket similar to the one found in Bcl-xL where Bak binds 415 . This was observed through crystal structures of the protein bound to BH3 peptides 414 To elucidate this structure, the author's used a truncated form of Bfl-1, lacking the 20 amino acids of the C-terminal 9 helix, and mutated hydrophobic residues expected to be solvent exposed to hydrophilic residues (P104K, C113S). These mutations were far away from the BH3 binding site.…”

Section: The Structure Of Bfl-1mentioning

confidence: 99%

“…Alternately, a conditional transgene-driven shRNA was used which knock-downed A1-a, A1-b and A1-d, although with different efficiencies in different cell types, being much more successful in thymocytes than mature lymphocytes and resulting in no obvious phenotype 421 . The mouse and human proteins contain 72% homology, and although the BH4 domain is present in Bfl-1 but completely lacking in the murine A1 protein, the proteins display very similar structures 414,416 . A splice variant of human Bfl-1 has also been distinguished called Bfl-1 Short (Bfl-1S).…”

Section: The Structure Of Bfl-1mentioning

confidence: 99%

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Role of the pro-survival molecule Bfl-1 in melanoma

Hind

Carter

Harris

et al. 2015

The International Journal of Biochemistry & Cell Biology

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Melanoma, the cancer derived from the melanocytes of the skin, is becoming an ever more common cancer in the ageing population of the developed world and displays an intrinsic resistance to current therapies. This chemo resistance makes metastatic melanoma an extremely difficult cancer to treat leading to a 5 year survival rate of just 20%. As such, it is important to unearth new potential drug targets to increase the prospects for melanoma patients.Bfl-1 is a pro-survival protein of the Bcl-2 family of apoptosis regulating proteins. It has been found to be over-expressed in a small subset of chemo resistant tumours, including melanoma. Accordingly, I characterised the molecule in melanoma cells and determined its role in protecting these cells from chemotherapy-induced apoptosis. I elucidated the expression profile and half-lives of the protein and mRNA across a panel of melanoma cell-lines and healthy melanocytes. I also confirmed, using pathway specific inhibitors, that Bfl-1 was transcriptionally regulated by the NFB pathway and concluded through pulsechase experiments that Bfl-1 protein was degraded, at least in part, by the proteasome. Subcellular fractionation and indirect immunofluorescence techniques elucidated that Bfl-1 was located mostly at the mitochondria in both resting and apoptotic cells, with a diffuse level present throughout the cytoplasm. As well as characterising the protein in both melanoma cells and healthy primary melanocytes, I determined its role in the resistance of these cells to apoptosis. Over-expressed Bfl-1 was found to protect melanoma cells from apoptosis caused by a range of chemotherapeutic agents in A375 melanoma cells, which naturally expressed very low levels of Bfl-1. Further, knock down of Bfl-1 using siRNA technology in melanoma cells revealed the dependence of these cells on Bfl-1 for their resistance to certain chemotherapeutic agents currently used in melanoma treatment, including the MEK inhibitor PD901. All together, this research contributes to our understanding of Bfl-1 and highlights it as a potentially important therapeutic target in metastatic melanoma.

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“…The threshold cutoff we used was 3 A to identify only true positives (less sensitive but more specific) (27). We used the structure of the Bcl-2/Bcl-X(L) chimera (PDB code 1GJH) (20) and Bfl-1, excluding the BH3 peptide from BIM (PDB code 2VM6) (28) to calculate the surface-exposed area by residue, considering buried all residues that are 5% exposed or less (29).…”

Section: Methodsmentioning

confidence: 99%

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins

Godoi

Wilkie-Grantham

Hishiki

et al. 2016

Journal of Biological Chemistry

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B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of crosstalk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.In metazoans, Bcl-2 5 family proteins regulate several cellular processes, most prominently apoptosis (programmed cell death) and also autophagy (1, 2). Based on primary amino acid sequence similarity, members of the Bcl-2 protein family share at least one of four modular components, termed Bcl-2 homology (BH) domains. Of these, BH3 has an amphipathic ␣-helical structure and plays a pivotal role in both the activation of proapoptotic and the suppression of anti-apoptotic members of the family. For example, the BH3 domains of Bim, Bid, and Puma trigger conformational changes in the executioners Bax and Bak, leading to their oligomerization and permeation of the outer mitochondrial membrane. Downstream consequences of Bax and Bak activation include release of cytochrome c and, ultimately, the activation of caspase-9 and other cell death proteases. Conversely, the BH3 domains of these and other BH3-carrying proteins bind anti-apoptotic Bcl-2 family proteins and inhibit their ability to prevent Bax/Bak oligomerization in the outer mitochondrial membrane.The BH3 domain of pro-apoptotic proteins operates analogous to a ligand to interact with a predominantly hydrophobic receptor-like binding crevice on the surfaces of anti-apoptotic Bcl-2 family proteins. In addition to regulating apoptosis, the BH3-binding pocket on anti-apoptotic Bcl-2 family proteins has been reported to bind a BH3-like domain in the autophagy protein Beclin-1, and this interaction has been shown to play an important role in regulating autophagy (3). Prior work has dissected the binding affinity of various BH3 peptides (ligands) to anti-apoptotic Bcl-2 proteins (receptors), and three-dimensional structural data are available for several of these pairs (reviewed in Ref. 4).Targeting the BH3-binding cleft of anti-apo...

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Completing the family portrait of the anti‐apoptotic Bcl‐2 proteins: Crystal structure of human Bfl‐1 in complex with Bim

Cited by 68 publications

References 33 publications

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

Role of the pro-survival molecule Bfl-1 in melanoma

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins

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